Cell culture medium

ABSTRACT

The present invention provides a basal cell culture medium and a feed medium with novel amino acid ratios and/or iron choline citrate as iron carrier that result in improved performance of mammalian cell culture processes, such as CHO cultivation and protein production processes, in particular in increased product titer (e.g. of monoclonal antibodies). Also provided are methods for culturing mammalian cells and producing a protein of interest using said basal cell culture medium and optionally feed medium. The invention also provides for a medium platform that comprises (i) the basal cell culture medium and (ii) the feed medium.

RELATED APPLICATION DISCLOSURE

This Application is a Divisional of U.S. patent application Ser. No.15/562,288, which is a U.S. national stage entry of InternationalApplication No. PCT/EP2016/057036, which claims priority to EuropeanPatent Application No. 15162228.9.

SEQUENCE DISCLOSURE

This application includes, as part of its disclosure, a “SequenceListing XML” pursuant to 37 C.F.R. § 1.831(a) which is submitted in XMLfile format via the USPTO patent electronic filing system in a filenamed “01-2861-US-2-2022-11-21-Sequence-Listing.xml”, created on Nov.11, 2022, and having a size of 8,951 bytes, which is hereby incorporatedby reference herein in its entirety.

BACKGROUND OF THE INVENTION Technical Field

The invention concerns the field of cell culture and recombinant proteinproduction in mammalian cells. It specifically concerns a novel basalcell culture medium as well as a novel feed medium for optimalproduction (e.g. titer) and performance (e.g. cell growth) in mammaliancell culture for products such as (poly)peptides, antibodies, antibodyfragments and antibody derived molecules using recombinant mammalianhost cells.

Background

The development of mammalian cell culture processes for large-scaleindustrial manufacturing of therapeutic proteins (e.g. monoclonalantibodies) began about 25 years ago. An efficient bioprocess for theproduction of biopharmaceuticals mainly requires (i) a high-producing,stable and regulatory-accepted (typically mammalian) cell line, (ii)optimal cell culture media to support cell growth and production indifferent (typically mammalian) host cells and in different cultivationsystems and process modes (for example at different scales and as e.g.batch, fed batch and perfusion processes), and (iii) an optimaltechnical bioprocess, characterized by e.g. optimal supply of oxygen byan adequate configuration of stirrers and gas supply, automated controlof all relevant process parameters to ensure consistent product quality,or process designs that can be scaled-up from small-scale processdevelopment (mL- to L-scale) to large-scale manufacturing (>2.000 L)without compromising performance and product quality.

In this context, cell culture media have a key role and have to fulfillthe complex nutritional requirements of mammalian cells cultivated insuspension in technical systems in contrast to their natural origin. Forexample, the most widely used cell line for biopharmaceutical productionwas originally derived from the Chinese hamster ovary (CHO cell).

In the past, serum was used as medium additive to provide nutrients orcarrier proteins that are typically not present in cell culture media,e.g. cholesterol and transferrin or factors for cell-substrateattachment (for example fibronectin), other hormones and growth factors,but also a protection of certain essential nutrients and binding oftoxic components within the culture medium. However, in cell culturemedia used for production of therapeutics, serum can potentiallyintroduce animal viruses and it may introduce other undesirablecontaminants into cell culture processes (for example antibiotics orproteases) due to the undefined origin of the raw material. Thesecompounds were successfully replaced and serum-free cell culture mediahave become industrial practice for process development and recombinantproduction of biopharmaceuticals. For example, CHO (Chinese hamsterovary) cells were serially propagated in serum-free medium containinghuman insulin as the only medium protein component (Keen and Rapson,Cytotechnology, October 17(3):153-63, 1995).

Another group of media components that is commonly used inbiopharmaceutical production are hydrolysates, either derived fromanimal origin or derived from plants. Due to the associated safety risk,hydrolysates from animal origin are removed from the process wheneverpossible. Hydrolysates usually contain a mixture of amino acids, smallpeptides, inorganic ions, trace elements, carbohydrates and vitamins andare widely used to enrich the culture medium with a variety of(essential) nutrients to increase overall growth and productivity.Another disadvantage, besides safety aspects, is the fact thathydrolysates are not chemically defined, thus the exact compositionbetween lots can change (lot-to-lot variability) and, for that reason,have a negative impact on process reproducibility. Since hydrolysatescontain many compounds and have a complex (in every detail unknown)composition they cannot easily be replaced without effecting cellculture performance (e.g. product yields). It is a persisting andunsolved challenge for bioprocess development to screen and subsequentlyreplace such undefined raw materials with chemical-defined componentsand maintaining consistent product quality and high product titers atthe same time. Not only the exact chemical composition needs to bedetermined, but also the exact concentrations of every component. Due tosafety aspects, today, mainly hydrolysates from plant origin are used(e.g. soy bean hydrolysates). However, the problem of undefinedcomposition remains.

Currently biopharmaceutical process development aims for chemicallydefined media (serum-free, animal component-free, chemically defined).This leads to a further reduced risk of contaminants (e.g. reduction oforganic materials, endotoxins, unknown metals and trace elements derivedfrom unknown nutrient sources) and also fosters an increased controlover all aspects of upstream and downstream processing with respect toconsistent raw materials without any risks related to safety andlot-to-lot variability. Only recently, “chemically defined” (note thatthere is no industry standard that clearly defines this term yet) mediabecame commercially available for mammalian cell culture but applicationin industrial manufacturing currently still is limited.

The exact composition of such “chemically defined” hydrolysates or mediasupplements are known (but typically only to the media supplier) so thatno undefined raw materials are in use. These chemically defined mediaare still complex and contain up to about 40-50 “key components”. Butthey are made up of different building blocks. To design such complex“chemically defined” media it is necessary to mimic plant or animalderived hydrolysates as close as possible which requires extensivefractionation of those hydrolysates followed by high-end analytics. Amajor challenge in this field is given by the tremendous diversity ofcompounds present in cell culture media.

Optimal design of high-performance cell culture media is furthercomplicated by the fact that many processes are performed in theso-called fed batch mode, i.e., a cell culture is inoculated in a basalstart medium and then (typically after about 0 to 3 days) a concentratedfeed medium is supplied to sustain growth and production when mediasubstrates get depleted due to cell growth. Providing all mediacompounds from the beginning (batch mode) results in suboptimal processperformance since then cells are overfed in the beginning (for exampleresulting in high formation of the unwanted by-product lactate which inturn has an adverse effect on cell growth and viability). The majorchallenge in this context is to design an optimal batch medium (basalcell culture medium) and an optimal fed batch medium (feed medium) thatperfectly match for optimal growth and production in a cell cultureprocess throughout the cultivation run time. Since viable cellconcentration, viability of the cell culture and nutritionalrequirements of a cell culture process significantly change over thetime course of a cultivation process (on an hourly to daily basis), thedesign of optimal batch and fed batch media (basal cell culture mediumand feed medium) is a demanding task. It is even more difficult sincetypically one feed medium is designed that provides the optimal solutionfor every single hour and every single day of a fermentation processwhich lasts in total up to about 2-3 weeks.

The state of the art in industrial mammalian cell culture medium designusing a “rational” approach has been summarized by Fletcher (FletcherT., BioProcess International 3(1), 30-36, 2005), and this approach canstill be considered as the state of the art concept for rational mediadesign in industrial practice. It is pointed out that the complexity ofrational medium design is not only given by the fact that manycomponents are involved but also that the specific concentrations andthe complex interactions of media compounds need to be considered.

According to Fletcher three basic approaches exist in medium design.These are i) (single) component titration (experiments to define a doseresponse e.g. on titer), ii) media blending (simply blend existing(complex) media and identify the best blend), iii) spent media analysis(describe nutrient depletion by chemical analysis of spent medium vs.fresh medium; note that specific metabolic needs on a cell basis are notconsidered in this approach), iv) automated screening (robotic fluidhandling with strong focus on throughput e.g. in multi-well plates).None of these methods is best in every way, and each has its ownparticular weaknesses according to Fletcher. For example, i) componenttitration causes immense amounts of samples to be analyzed which is notfeasible in industrial practice for many reasons (e.g. capacity, costs,resources), ii) media blending leads to improved throughput but thisapproach is poorly instructive and very limited in scope, iii) spentmedia analysis can provide important information how culture chemistrychanges over time, but it cannot provide a complete picture of the cellculture requirements based on the fact that typically spent mediaanalysis focuses only on a quite limited number of components (notethat, for example, a complete amino acid analysis of all 20 amino acidsis typically not performed and only the two most important amino acidsglutamine and glutamate are routinely measured), iv) automated screeningincreases throughput by minimizing the cultivation system but, in turn,has adverse effects on (correctly) modeling a large-scale process sincesuch miniaturized systems fail to correctly predict cell cultureperformance in the large-scale. Hence, Fletcher concluded that realrational media design can be described as multidimensional approach.Instead of relying on a single technique, rational media design makesuse of several complementary methods, namely DoE (Design of experiments)and full factorial designs that capture the complex interactions ofmultiple components and use various statistical tools. Although thisconcept integrates previous media design concepts and applies advancedDoE approaches for optimal design of experiments, it clearly lacks thecell-specific requirements, i.e. the cellular perspective of nutritionalsupply and cellular metabolism. Hence, there is still a need forimproved cell culture media.

Cell Culture Media:

In mammalian cell cultivation, cell culture media can comprise up toabout 100 compounds and more. For example, carbohydrates (e.g. forgeneration of energy by catabolic reactions or as building blocks byanaplerotic reactions), amino acids (e.g. building blocks for cellularprotein and product in case of therapeutic protein production), lipidsand/or fatty acids (e.g. for cellular membrane synthesis), DNA and RNA(e.g. for growth and cellular mitosis and meiosis), vitamins (e.g. asco-factors for enzymatic reactions), trace elements, different salts,growth factors, carriers and transporters etc. These components orcompound groups are required to fulfill the complex nutritionalrequirements of mammalian cells in a technical cultivation environment.There exist classical cell culture media such as DMEM (Dulbecco'sModified Eagle's Medium) where all components and all concentrations arepublished. Development of such cell culture media go back to the late1950s and are comprehensively described in the academic literature.Another example is Ham's F12 (Ham's Nutrient Mixture F12) that wasdeveloped in the 1970s, or mixtures/modifications of such classical cellculture such as DMEM:F12 (Dulbecco's Modified Eagle's Medium/Ham'sNutrient Mixture F12) that were developed in the 1970s and 1980s.Another widely used cell culture medium with known composition andconcentrations is RPMI. RPMI was developed in the 1970s by Moore et al.at the Roswell Park Memorial Institute (hence the acronym RPMI).Different variants are used in animal cell culture, for exampleRPMI-1640. Although many of these classical media were developed decadesago, these formulations still form the basis for much of the cellculture research occurring today and represent state of the art inanimal cell culture for media with completely known composition andcompletely known concentrations for each compound. All of these mediaare commercially available and can be obtained from suppliers (e.g. fromSigma-Aldrich).

Due to the increasing business in biopharmaceuticals, commercial mediasuppliers developed own cell culture media for use in mammalian cellculture over the past years.

However, in contrast to classical cell culture media, the exactformulations of such commercial cell culture media are proprietary tothe vendors. For this reason, such commercial media cannot be used as areference and starting point for rational media design since the exactformulation is not known (even for the major compounds such as aminoacids). For example, the commercially available medium ActiCHO (by PAA)consisting of a basal medium (ActiCHO P) and a feed medium (ActiCHO FeedA+B) is chemically defined according to supplier definition (only singlechemicals, free of animal derived substances, growth factors, peptides,and peptones). But the exact formulation is proprietary. The two feedsconsist of concentrated amino acids, vitamins, salts trace elements andcarbon source (Feed A) and selected amino acids in concentrated form(Feed B). Another example is Ex-Cell CD CHO (SAFC Biosciences). Thismedium is animal component free, chemically defined according to SAFC,serum-free, and formulation is also proprietary. A third example mediumthat is widely used in mammalian cell culture using CHO is CD CHO (Lifetechnologies). This medium is protein free, serum-free, and chemicallydefined according to Life technologies. It does not containproteins/peptides of animal, plant or synthetic origin or undefinedlysates/hydrolysates. Again formulation is proprietary. This CD CHObasal medium can be combined with feed media named Efficient Feed A, B,and C. Also for the feeds the formulation is proprietary. The feeds areanimal origin-free and the components are contained in higherconcentrations. The feeds are chemically defined. No proteins, nolipids, no growth factors, no hydrolysates and no components of unknowncomposition are used. It contains a carbon source, concentrated aminoacids, vitamins and trace elements. Another feed that is commerciallyavailable can be obtained by Thermo Fisher, named Cell Boost 1-6. Again,the formulation is proprietary. It is chemically defined according toThermo Fisher, protein free, and animal derived components free. CellBoost 1 and 2 contain amino acids, vitamins, and glucose. Cell Boost 3contains amino acids, vitamins, glucose, and trace elements. Cell Boost4 contains amino acids, vitamins, glucose, trace elements, and growthfactors. And Cell Boost 5 and 6 contain amino acids, vitamins, glucose,trace elements, growth factors, lipids, and cholesterol.

Amino Acids

Amino acids have an essential role for protein synthesis, both forcellular protein and for the production of the product in case ofrecombinant proteins or protein derived substances. For examples,proteins are synthesized by the cellular machinery from single aminoacids molecules to form larger proteins or protein complexes. Inmammalian cell cultivation the essential amino acids need to be providedwith the cell culture medium, since mammalian cells are not able tosynthesize essential amino acids from other precursors and buildingblocks. Amino acids are also biochemically important because thesemolecules have two functional groups (amino group and an acidic group)which enables them to interact with other biological molecules. Forthese reasons cell culture media containing amino acids are often alsosupplemented with a variety of (defined and undefined) small peptides,hydrolysates, proteins and protein mixtures from different origins(animal derived, plant derived or chemically defined).

In the context of the present invention it was found that specific aminoacid compositions and novel amino acid ratios both in the (basal) cellculture medium and in the feed medium significantly increase finalproduct titers. This new amino acid composition and amino acid ratiossignificantly differ from the state of the art of commercially availablecell culture media (e.g. RPMI, DMEM:F12 1:1) and provide higher producttiters.

Iron and Iron Carrier

Iron is an essential ingredient in mammalian cell culture media (i) as atrace element and (ii) as a transferrin replacement (e.g. iron as ironchelators). Transferrin is typically derived from plasma. This compoundis typically supplied as a lyophilized powder of human transferrin whichis partially iron-saturated. Transferrin is a glycoprotein withhomologous N-terminal and C-terminal iron-binding domains and is relatedto several other iron-binding proteins including lactoferrin,melanotransferrin, and ovotransferrin. Transferrin is commerciallyavailable for use in animal cell culture (e.g. by Sigma-Aldrich, CASnumber 11096-37-0). There exist several other iron compounds that areused as transferrin replacement. These exist in II/III forms, as varioussalts, as hydrated/dehydrated forms. Examples are iron (III) phosphate,iron (III) pyrophosphate, iron (III) nitrate, iron (II) sulfate, iron(III) chloride, iron (II) lactate, ferric (III) citrate, ammonium ferric(III) citrate, iron-dextran, or ethylenediaminetetraacetic acid ferricsodium salt.

We identified iron choline citrate (iron/ferric choline citrate,CAS-Number 1336-80-7, molecular weight Mw=991.5 g/mol+/−49.57 g/mol dueto 5% crystal water content, iron complex with iron content of about10.2-12.4%, molecule ratio for iron:choline:citrate of 2:3:3, moleculeformula C₃₃H₅₇Fe₂N₃O₂₄). However, other suitable iron choline citratecomplexes are known such as iron:choline:citrate at a ratio of 1:1:1,molecular weight of Mw=348.11 g/mol. Compared to state of the art ironsources used in commercially available cell culture media such as ironphosphate, iron pyrophosphate or iron citrate, the usage of iron cholinecitrate contributes to significantly higher product titers. This effectalso depends on the iron choline citrate concentration in the media.

SUMMARY OF THE INVENTION

The present invention provides a basal cell culture medium and a feedmedium with novel amino acid ratios and/or iron choline citrate as ironcarrier that improve the performance of mammalian cell cultureprocesses, such as CHO cultivation and protein production processes, inparticular product titers (e.g., monoclonal antibody (mAb) titres). Alsoprovided are methods for culturing mammalian cells and producing aprotein of interest using said basal cell culture medium and/or feedmedium. The invention also provides for a medium platform that comprises(i) the basal cell culture medium and (ii) the feed medium. Preferably,both the (basal) cell culture medium and the feed medium are chemicallydefined.

In one aspect the invention relates to a basal cell culture medium forculturing mammalian cells comprising the following amino acids at amolar ratio relative to isoleucine (mM/mM) of: L-leucine/L-isoleucine ofabout 1.2-2.2, L-phenylalanine/L-isoleucine of about 0.5-0.9,L-tyrosine/L-isoleucine of about 1.5-2.7, L-threonine/I-isoleucine ofabout 1.0-1.9, and L-valine/L-isoleucine of about 1.0-1.9, wherein thebasal cell culture medium has a total amino acid content of about 25 to150 mM. In one embodiment the basal cell culture medium furthercomprises L-lysine at a molar ratio relative to isoleucine of about1.6-2.9 (mM/mM). The basal cell culture medium may further comprise atleast one of the following amino acids at a molar ratio relative toisoleucine (mM/mM) of: L-tryptophan/L-isoleucine of about 0.3-0.5,L-proline/L-isoleucine of about 1.6-3.0; or L-methionine/L-isoleucine ofabout 0.4-0.7. Preferably the basal cell culture medium comprisesL-tryptophan, L-proline and L-methionine each at said molar ratios asdefined above. The basal cell culture medium of the invention is aserum-free medium, preferably a chemically defined medium or achemically defined and protein-free medium. In one embodiment the basalcell culture medium additionally comprises iron choline citrate at aconcentration of about 0.1 to 5.0 mM, about 0.2 to 2.0 mM, about 0.2 to1.0 mM or about 0.4 to 1.0 mM. In certain embodiments the basal cellculture medium has a total amino acid content of about 30 to about 130,preferably about 35 to about 120, more preferably about 40 to about 100mM.

The present invention also relates to a basal cell culture medium forculturing mammalian cells comprising iron choline citrate at aconcentration of about 0.1 to 5.0 mM, about 0.2 to 2.0 mM, about 0.2 to1.0 mM or about 0.4 to 1.0 mM.

In another aspect the present invention relates to a feed medium forculturing mammalian cells comprising the following amino acids at amolar ratio relative to isoleucine (mM/mM) of: L-leucine/L-isoleucine ofabout 2.3-4.2, L-phenylalanine/L-isoleucine of about 0.6-1.1,L-threonine/I-isoleucine of about 1.3-2.4, and L-valine/L-isoleucine ofabout 1.1-2.0, wherein the feed medium has a total amino acid content ofabout 100 to 1000 mM. In one embodiment of the present invention thefeed medium further comprises the following amino acids at a molar ratiorelative to isoleucine (mM/mM) of: L-tyrosine/L-isoleucine of about0.6-1.1, and/or L-lysine/L-isoleucine of about 1.1-2.1. The feed mediumaccording to the invention may further comprise at least one of thefollowing amino acids at a molar ratio relative to isoleucine (mM/mM)of: L-tryptophan/L-isoleucine of about 0.3-0.6, L-proline/L-isoleucineof about 0.9-1.8; or L-methionine/L-isoleucine of about 0.4-0.8.Preferably the feed medium comprises L-tryptophan, L-proline andL-methionine each at said molar ratios as defined above. The feed mediumis typically a concentrated feed medium. Preferably the feed medium ofthe invention is a serum-free medium, more preferably a chemicallydefined medium or a chemically defined and protein-free medium. In oneembodiment the feed medium additionally comprises iron choline citrateat a concentration of about 0.4 to 5 mM, about 0.4 to 1.0 mM or about0.5 to 1.0 mM, preferably about 0.5 to 0.6 mM. In one embodiment thefeed medium is characterized by a low salt content, preferably a lowsalt content of about 100 mM or less and more preferably about 50 mM orless. In certain embodiments the feed medium of the invention has atotal amino acid content of about 200 to about 900, preferably about 300to about 800, more preferably about 400 to about 700 mM.

The present invention also relates to a feed medium for culturingmammalian cells comprising iron choline citrate at a concentration ofabout 0.4 to 5 mM, about 0.4 to 1.0 mM or about 0.5 to 1.0 mM,preferably about 0.5 to 0.6 mM.

In a related aspect the invention relates to a medium platform forculturing mammalian cells comprising the basal cell culture medium ofthe invention and the feed medium of the invention as described herein.

The basal cell culture medium and the feed medium of the invention areparticularly suitable for culturing rodent or human cells, wherein therodent cell is preferably a Chinese hamster ovary (CHO) cell such as aCHO-K1 cell, a CHO-DG44 cell, a CHO-DUKX B11 cell or a CHO glutaminesynthetase (GS) deficient cell, most preferably the cell is a CHO-DG44or a CHO GS deficient cell.

In yet another aspect the invention relates to a method of generating abasal cell culture medium comprising: a) providing a basal cell culturemedium, and b) adding amino acids at or adjusting the amino acid ratiosto the final molar ratio according to the invention. The method mayfurther comprise a step of adding or adjusting as an iron source ironcholine citrate at a concentration of about 0.1 to 5.0 mM, about 0.2 to2.0 mM, about 0.2 to 1.0 mM, or about 0.4 to 1.0 mM.

In yet another aspect the invention relates to a method of generating afeed medium comprising: providing a feed medium, and adding amino acidsat or adjusting the amino acid ratios to the final molar ratiosaccording to the invention. The method may further comprise a step ofadding or adjusting as an iron source iron choline citrate at aconcentration of about 0.4 to 5 mM, about 0.4 to 1.0 mM, or about 0.5 to1 mM, preferably about 0.5 to 0.6 mM.

The invention further relates to a method of culturing a mammalian cellcomprising the following steps: a) providing mammalian cells, b)culturing the cells in the basal cell culture medium of the invention,and c) optionally adding the feed medium of the invention to the basalcell culture medium; wherein the cells are cultured under conditionsthat allow the cells to proliferate.

The invention also relates to a method of producing a protein ofinterest comprising the following steps: a) providing mammalian cellscomprising a gene of interest encoding the protein of interest, b)culturing the cells in the basal cell culture medium of the invention,and c) optionally adding the feed medium of the invention to the basalcell culture medium, and d) optionally separating and/or isolatingand/or purifying said protein of interest from the cell culture; whereinthe cells are cultured under conditions that allow expression of theprotein of interest. The protein of interest may be a secreted protein,preferably the protein of interest is an antibody or Fc-fusion protein.

The mammalian cell used in any of the methods of the invention may be arodent or human cell, preferably the rodent cell is a Chinese hamsterovary (CHO) cell such as a CHO-K1 cell, a CHO-DG44 cell, a DuxB11 cellor a CHO GS deficient cell, most preferably the cell is a CHO-DG44 or aCHO GS deficient cell. The feed medium used in any of the methods of theinvention is to be added to the cells cultured in the basal cell culturemedium, wherein (a) the feed medium is added at about 10-50 ml/L/daybased on the culture starting volume to the basal cell culture medium,(b) the feed medium is added starting on day 0, 1, 2 or 3, and/or (c)the feed medium is added continuously, or as a bolus several times aday, two times a day, once per day, every second day or every third day.

In yet another aspect the invention relates to a kit of parts comprisingthe basal cell culture medium of the invention and/or the feed medium ofthe invention, and optionally a mammalian cell.

The invention further relates to a use of the basal cell culture mediumof the invention for producing a protein comprising culturing mammaliancells that produce a protein of interest in said medium for a period oftime and conditions suitable for cell growth and protein production,harvesting the protein of interest and recovering the protein from theculture medium or cell lysate. The use may further comprise feeding thecells with the feed medium of the invention during said culture period.

The invention also relates to a use of the feed medium of the inventionfor producing a protein comprising culturing mammalian cells thatproduce the protein of interest in the basal cell culture medium of theinvention for a period of time and conditions suitable for cell growthand protein production, feeding the cells with said feed medium,harvesting the protein of interest and recovering the protein from theculture medium.

Also referred to is a use of iron choline citrate as iron carrier in amammalian cell culture medium, wherein the iron choline citrate ispresent in the mammalian cell culture medium at a concentration of about0.2 to 2.0 mM.

DESCRIPTION OF THE FIGURES

FIG. 1 : RPMI based basal medium with and without optimized amino acidadjustment in a batch experiment at different total amino acidconcentrations.

(A-D): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 4.1 and medium 5.0 without and with optimized amino acid ratiosin duplicates (N=2) with a total amino acid concentration of 44 mM, ●culture in medium 5.0, with optimized amino acid ratios (44 mM), ▪culture in medium 4.1, without optimized amino acid ratios (44 mM).Shown are (A) viable cell concentration [1×10⁵ cell/mL] CHO2 (CHO-DG44)Rituximab cells, (B) viability [%] CHO2 (CHO-DG44) Rituximab cells and(C) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab, (D) Lactateconcentration [g/L] CHO2 (CHO-DG44) Rituximab.

(E-H): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 4.2 and medium 5.1 without and with optimized amino acid ratiosin duplicates (N=2) with a total amino acid concentration of 66 mM, ●culture in medium 5.1, with optimized amino acid ratios (66 mM), ▪culture in medium 4.2, without optimized amino acid ratios (66 mM).Shown are (E) viable cell concentration [1×10⁵ cell/mL] CHO2 (CHO-DG44)Rituximab cells, (F) viability [%] CHO2 (CHO-DG44) Rituximab cells and(G) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab, (H) Lactateconcentration [g/L] CHO2 (CHO-DG44) Rituximab.

(I): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI based medium4.3 and medium 5.2 without and with optimized amino acid ratios induplicates (N=2) with a total amino acid concentration of 22 or 36 mM, ▪culture in medium 5.2, with optimized amino acid ratios (22 mM),

culture in medium 4.3, without optimized amino acid ratios (22 mM), +culture in medium 4.0, without optimized amino acid ratios (36 mM).Shown is (I) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab.

(J): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI based medium4.2 and medium 5.2 without and with optimized amino acid ratios induplicates (N=2) with a total amino acid concentration of 22 and 66 mM,▪ culture in medium 5.2, with optimized amino acid ratios (22 mM), ●culture in medium 4.2, without optimized amino acid ratios (66 mM).Shown is (J) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab.

FIG. 2 : RPMI based basal medium with a variation of single amino acidsby −20% or −40% based on optimized amino acid ratios in a batchexperiment.

(A-D): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 5.3 (with all amino acids at optimized ratios, control) andmedium 5.3.1 (only a single amino acid is modified by −20%),

culture in medium 5.3.1 (L-leucine −20%), + culture in medium 5.3.1(L-valine −20%), □ culture in medium 5.3.1 (L-phenylalanine −20%), ▪culture in medium 5.3 (with all amino acids, control), ● culture inmedium 5.3.1 (L-arginine −20%), ♦ culture in medium 5.3.1 (L-asparagine−20%), ▴ culture in medium 5.3.1 (L-aspartic acid −20%), ▾ culture inmedium 5.3.1 (L-histidine −20%),

culture in medium 5.3.1 (L-lysine −20%), × culture in medium 5.3.1(L-methionine −20%), ◯ culture in medium 5.3.1 (L-proline −20%), ⋄culture in medium 5.3.1 (L-serine −20%), Δ culture in medium 5.3.1(L-threonine −20%), ∇ culture in medium 5.3.1 (L-tryptophan −20%),(triangle left, empty) culture in medium 5.3.1 (L-tyrosine −20%). Shownare (A) viable cell concentration [1×10⁵ cell/mL] CHO2 (CHO-DG44)Rituximab cells, (B) viability [%] CHO2 (CHO-DG44) Rituximab cells, (C)product concentration [mg/L] CHO2 (CHO-DG44) Rituximab, (D) lactateconcentration [mg/L] CHO2 (CHO-DG44) Rituximab, controls were performedin triplicate (N=3) and test runs in duplicates (N=2).

(E-G): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 5.3 (with all amino acids at optimized ratios, control) andmedium 5.3.1 (only a single amino acid, e.g., L-leucine, L-valine orL-phenylalanine, is modified by −20%), ▪ culture in medium 5.3 (with allamino acids, control), □ culture in medium 5.3.1 (L-phenylalanine −20%),

culture in medium 5.3.1 (L-leucine −20%), + culture in medium 5.3.1(L-valine by −20%), shown are (E-G) product concentration [mg/L] CHO2(CHO-DG44) Rituximab, controls were performed in triplicate (N=3) andtest runs in duplicates (N=2).

(H-K): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 5.3 (with all amino acids at optimized ratios, control) andmedium 5.3.1 (only a single amino acid, e.g., L-arginine, L-asparagine,L-aspartic acid, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine or L-valine, is reduced by −40%), ▪ culture inmedium 5.3 (with all amino acids, control), ● culture in medium 5.3.1(L-arginine −40%), ♦ culture in medium 5.3.1 (L-asparagine −40%), ▴culture in medium 5.3.1 (L-aspartic acid −40%), ▾ culture in medium5.3.1 (L-histidine −40%),

culture in medium 5.3.1 (L-isoleucine-40%),

culture in medium 5.3.1 (L-leucine −40%), + culture in medium 5.3.1(L-lysine −40%), □ culture in medium 5.3.1 (L-methionine −40%), ◯culture in medium 5.3.1 (L-phenylalanine −40%), ⋄ culture in medium5.3.1 (L-proline −40%), Δ culture in medium 5.3.1 (L-serine −40%), ∇culture in medium 5.3.1 (L-threonine −40%), (triangle left, empty)culture in medium 5.3.1 (L-tryptophan −40%), (triangle right, empty)culture in medium 5.3.1 (L-tyrosine −40%), × culture in medium 5.3.1(L-valine −40%), shown are (H) viable cell concentration [1×10⁵ cell/mL]CHO2 (CHO-DG44) Rituximab cells, (I) viability [%] CHO2 (CHO-DG44)Rituximab cells, (J) product concentration [mg/L] CHO2 (CHO-DG44)Rituximab, (K) lactate concentration [mg/L] CHO2 (CHO-DG44) Rituximab,controls were performed in triplicate (N=3) and test runs in duplicates(N=2).

(L-O): CHO2 (CHO-DG44) Rituximab cells were cultured in RPMI basedmedium 5.3 (with all amino acids at optimized ratios, control) andmedium 5.3.1 (only a single amino acid, e.g., L-phenylalanine, L-valine,L-leucine, L-threonine or L-isoleucine, is reduced by −40%), ▪ culturein medium 5.3 (with all amino acids, control), ◯ culture in medium 5.3.1(L-phenylalanine −40%), × culture in medium 5.3.1 (L-valine −40%),

culture in medium 5.3.1 (L-leucine is reduced by −40%), ∇ culture inmedium 5.3.1 (L-threonine −40%),

culture in medium 5.3.1 (L-isoleucine −40%). Shown are (L) viable cellconcentration [1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (M)viability [%] CHO2 (CHO-DG44) Rituximab cells, (N) product concentration[mg/L] CHO2 (CHO-DG44) Rituximab, (O) lactate concentration [mg/L] CHO2(CHO-DG44) Rituximab, controls were performed in triplicate (N=3) andtest runs in duplicates (N=2).

FIG. 3 : Variation of a single amino acid by −40% based on optimizedamino acid ratios in basal medium in a batch experiment. (A-C): Cellswere cultivated in batch mode in medium 6.4.10-6.4.15 (only a singleamino acid, e.g., L-lysine, L-methionine, L-proline, L-tryptophan orL-tyrosine, or the two amino L-tyrosine and L-lysine is/are reduced by−40%) or in control medium 6.4.0.1 (with optimized amino acid ratios), ▪culture in medium 6.4.0.1 (with all amino acids, control), + culture inmedium 6.4.10 (L-tyrosine and L-lysine −40%), ♦ culture in medium 6.4.11(L-tyrosine −40%), □ culture in medium 6.4.12 (L-lysine −40%), ● culturein medium 6.4.13 (L-methionine −40%), ⋄ culture in medium 6.4.14(L-tryptophan −40%), (×) culture in medium 6.4.15 (L-proline −40%).Shown are (A) viable cell concentration [1×10⁵ cell/mL] CHO2 (CHO-DG44)Rituximab cells, (B) viability [%] CHO2 (CHO-DG44) Rituximab cells and(C) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab, allexperiments were performed in duplicates (N=2).

FIG. 4 : Effect of optimized medium and feed medium in a fed-batchexperiment at a standard or reduced feed rate.

(A-C): Effect of optimized basal medium and feed medium in a fed-batchexperiment at a standard feed rate. Cells were cultivated in basalmedium 6.2 (with optimized amino acid ratios), medium 6.3 (withoutoptimized amino acid ratios), feed medium 6.2 (with optimized amino acidratios) and feed medium 6.3 (without optimized amino acid ratios). CHO2(CHO-DG44) Rituximab cells were cultured in various combinations ofbasal medium and feed medium, ▪ culture in basal medium 6.2 (withoptimized amino acid ratios) and feed medium 6.2 (with optimized aminoacid ratios), ● culture in basal medium 6.2 (with optimized amino acidratios) and feed medium 6.3 (without optimized amino acid ratios), ♦culture in basal medium 6.3 (without optimized amino acid ratios) andfeed medium 6.2 (with optimized amino acid ratios), ▴ culture in basalmedium 6.3 (without optimized amino acid ratios) and feed medium 6.3(without optimized amino acid ratios, shown are (A) viable cellconcentration [1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (B)viability [%] CHO2 (CHO-DG44) Rituximab cells and (C) productconcentration [mg/L] CHO2 (CHO-DG44) Rituximab, all experiments wereperformed in duplicates (N=2).

(D-F): Effect of optimized basal medium and feed medium at a reducedfeed rate. Cells were cultivated in medium 6.2 (with optimized aminoacid ratios), medium 6.3 (without optimized amino acid ratios), feedmedium 6.2 (with optimized amino acid ratios) and feed medium 6.3(without optimized amino acid ratios). CHO2 (CHO-DG44) Rituximab cellswere cultured in various combinations of basal medium and feed medium.The feed rate for all cultures was reduced to provoke a strong responseof the cultures, ▪ culture in basal medium 6.2 (with optimized aminoacid ratios) and feed medium 6.2 (with optimized amino acid ratios andreduced feed rate, ● culture in basal medium 6.2 (with optimized aminoacid ratios) and feed medium 6.3 (without optimized amino acid ratios),♦ culture in basal medium 6.3 (without optimized amino acid ratios) andfeed medium 6.2 (with optimized amino acid ratios), ▴ culture in basalmedium 6.3 (without optimized amino acid ratios) and feed medium 6.3(without optimized amino acid ratios). Shown are (D) viable cellconcentration [1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (E)viability [%] CHO2 (CHO-DG44) Rituximab cells and (F) productconcentration [mg/L] CHO2 (CHO-DG44) Rituximab, all experiments wereperformed in duplicates (N=2).

(G-J): Effect of optimized medium and feed medium in 2-L scale. Cellswere cultivated in a fully controlled 2-L system in medium 6.2 (withoptimized amino acid ratios), medium 6.3 (without optimized amino acidratios), feed medium 6.2 (with optimized amino acid ratios) and feedmedium 6.3 (without optimized amino acid ratios). CHO2 (CHO-DG44)Rituximab cells were cultured in various combinations of optimized basalmedium and feed medium, ▪ culture in basal medium 6.2 (with optimizedamino acid ratios) and feed medium 6.2 (with optimized amino acidratios, ● culture in basal medium 6.2 (with optimized amino acid ratios)and feed medium 6.3 (without optimized amino acid ratios), ♦ culture inbasal medium 6.3 (without optimized amino acid ratios) and feed medium6.2 (with optimized amino acid ratios), shown are (G) viable cellconcentration [1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (H)viability [%] CHO2 (CHO-DG44) Rituximab cells, (I) product concentration[mg/L] CHO2 (CHO-DG44) Rituximab, (J) lactate concentration [g/L] CHO2(CHO-DG44) Rituximab, all experiments were performed in duplicates(N=2).

(K-M): Effect of optimized RPMI medium and RPMI feed medium. Cells werecultivated in RPMI basal medium 3.1 (without optimized amino acidratios), RPMI medium 3.9 (with optimized amino acid ratios), RPMI feedmedium-2 (without optimized amino acid ratios) and RPMI feed medium-3(with optimized amino acid ratios). CHO2 (CHO-DG44) Rituximab cells werecultured in various combinations of optimized basal medium and feedmedium, ▪ culture in RPMI medium 3.9 (with optimized amino acid ratios)and RPMI feed medium-3 (with optimized amino acid ratios, ● culture inRPMI medium 3.9 (with optimized amino acid ratios) and RPMI feedmedium-2 (without optimized amino acid ratios), ♦ culture in RPMI medium3.1 (without optimized amino acid ratios) and RPMI feed medium-3 (withoptimized amino acid ratios), ▴ culture in RPMI medium 3.1 (withoutoptimized amino acid ratios) and RPMI feed medium-2 (without optimizedamino acid ratios), shown are (K) viable cell concentration [1×10⁵cell/mL] CHO2 (CHO-DG44) Rituximab cells, (L) viability [%] CHO2(CHO-DG44) Rituximab cells, (M) product concentration [mg/L] CHO2(CHO-DG44) Rituximab, all experiments were performed in duplicates(N=2).

(N-P): Effect of optimized RPMI medium and RPMI feed medium compared tobasal and feed medium without optimized amino acid (AA) ratios or spendmedia optimized AA ratios. Cells were cultivated in RPMI medium 3.1(without optimized amino acid ratios), RPMI medium 3.9 (with optimizedamino acid ratios), RPMI feed medium-2 (without optimized amino acidratios) and RPMI feed medium-3 (with optimized amino acid ratios), RPMImedium 3.5 (with spend media supplemented AAs), RPMI feed medium 3.5(with spend media supplemented AAs). CHO2 (CHO-DG44) Rituximab cellswere cultured in various combinations of RPMI basal medium and RPMI feedmedium, ▪ culture in RPMI medium 3.9 (with optimized amino acid ratios)and RPMI feed medium-3 (with optimized amino acid ratios, ● culture inRPMI medium 3.9 (with optimized amino acid ratios) and RPMI feedmedium-2 (without optimized amino acid ratios, ♦ culture in RPMI medium3.1 (without optimized amino acid ratios) and RPMI feed medium-2(without optimized amino acid ratios), ▴ culture in RPMI medium 3.1(without optimized amino acid ratios) and RPMI feed medium-3 (withoptimized amino acid ratios, ◯ culture in RPMI medium 3.5 (with spendmedia supplemented AAs) and RPMI feed medium-2 (without optimized aminoacid ratios), □ culture in RPMI medium 3.5 (with spend mediasupplemented AAs) and RPMI feed medium-3.5 (with spend mediasupplemented AAs), shown are (N) viable cell concentration [1×10⁵cell/mL] CHO2 (CHO-DG44) Rituximab cells, (0) viability [%] CHO2(CHO-DG44) Rituximab cells and (P) product concentration [mg/L] CHO2(CHO-DG44) Rituximab, all experiments were performed in duplicates(N=2).

FIG. 5 : Variation of 5 or 7 amino acids by +/−20% or +/−40% in a fedbatch experiment.

(A-C): Variation of 5 amino acids by +/−40% based on novel amino acidratios in optimized medium and feed. The amino acids L-phenylalanine,L-valine, L-leucine, L-threonine, L-isoleucine were varied by +/−40% ina positive or negative alternating mode (capital or non-capital AAletters) compared to control (with optimized amino acid ratios). CHO2(CHO-DG44) Rituximab cells were cultured in fed-batch in medium 6.4.0.1(with optimized amino acid ratios, control), basal medium 6.4.3 (5 aminoacids PHE, val, LEU, thr, ILE varied by +/−40%, positive), basal medium6.4.4 (5 amino acids phe, VAL, leu, THR, ile varied by +/−40%,negative), feed medium 6.4 (with optimized amino acid ratios, control),feed medium 6.4.3 (5 amino acids PHE, val, LEU, thr, ILE varied by+/−40%, positive), feed medium 6.4.4 (5 amino acids phe, VAL, leu, THR,ile varied by +/−40%, negative), ▪ culture in basal medium 6.4.0.1 andfeed medium 6.4 (with optimized amino acid ratios, control), × culturein basal medium 6.4.3 and feed medium 6.4.3 (5 amino acids PHE, val,LEU, thr, ILE varied by +/−40%, positive), ● culture in basal medium6.4.4 and feed medium 6.4.4 (5 amino acids phe, VAL, leu, THR, ilevaried by +/−40%, negative), shown are (A) viable cell concentration[1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (B) viability [%] CHO2(CHO-DG44) Rituximab cells and (C) product concentration [mg/L] CHO2(CHO-DG44) Rituximab, all experiments were performed in duplicates(N=2).

(D-F): Variation of 7 amino acids based on novel amino acid ratios inoptimized medium and feed. The amino acids L-phenylalanine, L-valine,L-leucine, L-threonine, L-isoleucine, L-tyrosine, L-lysine were variedby +/−40% in a positive or negative alternating mode (capital ornon-capital AA letters) compared to control (with optimized amino acidratios). CHO2 (CHO-DG44) Rituximab cells were cultured in fed-batch inmedium 6.4.0.1 (with optimized amino acid ratios, control), basal medium6.4.7 (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by +/−40%,positive), basal medium 6.4.8 (7 amino acids phe, VAL, leu, THR, ile,TYR, lys varied by +/−40%, negative), feed medium 6.4 (with optimizedamino acid ratios, control), feed medium 6.4.7 (7 amino acids PHE, val,LEU, thr, ILE, tyr, LYS varied by +/−40%, positive), feed medium 6.4.8(7 amino acids phe, VAL, leu, THR, ile, tyr, lys varied by +/−40%,negative), ▪ culture in basal medium 6.4.0.1 and feed medium 6.4 (withoptimized amino acid ratios, control), + culture in basal medium 6.4.7and feed medium 6.4.7 (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYSvaried by +/−40%, positive), ● culture in basal medium 6.4.8 (7 aminoacids phe, VAL, leu, THR, ile, TYR, lys varied by +/−40%, negative) andfeed medium 6.4.8 (7 amino acids phe, VAL, leu, THR, ile, tyr, lysvaried by +/−40%, negative), shown are (D) viable cell concentration[1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximab cells, (E) viability [%] CHO2(CHO-DG44) Rituximab cells, (F) product concentration [mg/L] CHO2(CHO-DG44) Rituximab, all experiments were performed in duplicates(N=2).

(G-H): Variation of 7 amino acids based on novel amino acid ratios inoptimized medium and feed. The amino acids L-phenylalanine, L-valine,L-leucine, L-threonine, L-isoleucine, L-tyrosine, L-lysine were variedby +/−20% and +/−40% in a positive or negative alternating mode atreduced feed rates (capital or non-capital AA letters) compared tocontrol (with optimized amino acid ratios). CHO2 (CHO-DG44) Rituximabcells were cultured in fed-batch in medium 6.4.0.1 and feed medium 6.4(with optimized amino acid ratios, for control standard feed rate andalso reduced feed rate), basal medium 6.4.5 (7 amino acids PHE, val,LEU, thr, ILE, tyr, LYS varied by +/−20%, positive), basal medium 6.4.7(7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by +/−40%,positive), basal medium 6.4.8 (7 amino acids phe, VAL, leu, THR, ile,TYR, lys varied by +/−40%, negative), feed medium 6.4 (with optimizedamino acid ratios, control), feed medium 6.4.5 (7 amino acids PHE, val,LEU, thr, ILE, tyr, LYS varied by +/−20%, positive), feed medium 6.4.7(7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by +/−40%,positive) and feed medium 6.4.8 (7 amino acids phe, VAL, leu, THR, ile,tyr, lys varied by +/−40%, negative) at reduced feed rate, ▪ culture inbasal medium 6.4.0.1 and feed medium 6.4 (with optimized amino acidratios, control) at standard feed rate, ● culture in basal medium6.4.0.1 and feed medium 6.4 (with optimized amino acid ratios, control)and reduced feed rate, + culture in basal medium 6.4.5 (7 amino acidsPHE, val, LEU, thr, ILE, tyr, LYS varied by +/−20%, positive) and feedmedium 6.4.5 (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by+/−20%, positive), × culture in basal medium 6.4.7 (7 amino acids PHE,val, LEU, thr, ILE, tyr, LYS varied by +/−40%, positive) and feed medium6.4.7 (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by +/−40%,positive), ▴ culture in basal medium 6.4.8 (7 amino acids phe, VAL, leu,THR, ile, TYR, lys varied by +/−40%, negative) and feed medium 6.4.8 (7amino acids phe, VAL, leu, THR, ile, tyr, lys varied by +/−40%,negative), shown are (G) viable cell concentration [1×10⁵ cell/mL] CHO2(CHO-DG44) Rituximab cells, (H) viability [%] CHO2 (CHO-DG44) Rituximabcells, and (I) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab,all experiments were performed in duplicates (N=2).

FIG. 6 : Fed-Batch of CHO-DG44 derived cell lines producing differenttherapeutic molecules were cultivated in an optimized basal medium 6.2and feed medium 6.2 (with optimized amino acid ratios in basal mediumand feed medium, without hydroxyl-L-proline), ▾ Fc-fusion proteinproduced in CHO-DG44 cells, ▪ Rituximab (IgG1 kappa) antibody producedin CHO-DG44 cells (CHO2 (CHO-DG44) Rituximab),

mAb5/IgG1 kappa antibody produced in CHO-DG44 cells,

mAb6/IgG1 kappa produced in CHO-DG44 cells. Shown are (A) viable cellconcentration [1×10⁵ cell/mL], (B) viability [%] CHO-DG44, (C) productconcentration [mg/L] for mAb5/IgG1 and mAb6/IgG1, and (D) productconcentration [mg/L] Fc-fusion protein and Rituximab, all experimentswere performed in duplicates (N=2).

FIG. 7 : Comparison of iron choline citrate with other iron carrier atabout equimolar amounts.

(A, B) CHO2 (CHO-DG44) Rituximab cells were cultured in basal medium6.2a with the indicated iron carrier in the basal medium and feed medium6.2a without iron choline citrate in fed batch mode, ▪ culture in basalmedium 0.2 g/L iron choline citrate, ● culture in basal medium with 1.0g/L iron choline citrate; ♦ culture in basal medium with 2.0 g/L ironcholine citrate; ▴ culture in basal medium with 0.5 g/L iron pyrophosphate; + culture in basal medium with 0.8 g/L iron pyro phosphate,(filled star) culture in basal medium with 1.3 g/L iron pyro phosphate,▾ culture in basal medium with 0.3 g/L iron phosphate, (filled pentagon)culture in basal medium with 0.5 g/L iron phosphate,

culture in basal medium with 0.7 g/L iron phosphate. Shown are (A)viable cell concentration [1×10⁵ cell/mL] CHO2 (CHO-DG44) Rituximabcells, (B) product concentration [mg/L] CHO2 (CHO-DG44) Rituximab, allexperiments were performed in duplicates (N=2).

(C) Product concentration [mg/L] of CHO2 (CHO-DG44) Rituximab cellscultured in basal medium 6.2a with different concentrations of ironcholine citrate and feed medium 6.2a containing 0.56 g/l iron cholinecitrate in fed batch mode (N=2), ▪ culture in basal medium without ironcholine citrate, ♦ culture in basal medium with 0.2 g/L iron cholinecitrate; ▴ culture in basal medium with 0.4 g/L iron choline citrate; +culture in basal medium with 2.0 g/L iron choline citrate.

(D) Product concentration [mg/L] of CHO2 (CHO-DG44) Rituximab cellscultured in basal medium 6.2a and feed medium 6.2a with iron cholinecitrate or iron citrate at about equimolar amounts in fed batch mode(N=2), ▪ culture in basal medium without iron choline citrate and feedmedium with 0.56 g/l iron choline citrate, (filled star) culture inbasal medium with 0.2 g/L iron choline citrate and feed medium with 0.56g/l iron choline citrate; ▾ culture in basal medium with 0.1 g/L ironcitrate and feed medium with 0.25 g/l iron citrate.

FIG. 8 : Comparison of iron choline citrate with iron citrate at aboutequimolar amounts in RPMI based medium. Product concentration CHO2(CHO-DG44) Rituximab cells cultured in basal medium 3.1 with ironcholine citrate or iron citrate at about equimolar amounts and feedmedium 2 containing 0.25 g/l iron citrate in fed batch mode (N=2), ▪culture in basal medium without iron choline citrate, ● culture in basalmedium with 0.2 g/L iron choline citrate; + culture in basal medium with0.1 g/L iron citrate. Shown are product concentration [mg/L] CHO2(CHO-DG44) Rituximab comparing (A) 0.2 g/l iron choline citrate (●) with0.1 g/l iron citrate (+), (B) 0.4 g/l iron choline citrate (▴) with 0.2g/l iron citrate (pentagon) (C) 2 g/l iron choline citrate (

) with 1 g/l iron citrate (●) and (D) 0.2 g/l (●) and 2 g/l (

) iron choline citrate.

FIG. 9 : Comparison of iron choline citrate with iron citrate at aboutequimolar amounts in medium 6.2a or RPMI based medium in fed batch modein a 2 L bioreactor. CHO2 (CHO-DG44) Rituximab cells were cultured in(A-C) basal medium 6.2a with iron choline citrate or iron citrate andfeed medium 6.2a containing 0.56 g/l iron choline citrate (D) or inbasal medium 3.1 with iron choline citrate or iron citrate and feedmedium-2 containing 0.25 g/l iron citrate in fed batch mode (N=2), +culture in basal medium 6.2a with 0.2 g/l iron choline citrate and feedmedium 6.2a with 0.56 g/l iron choline citrate, (filled star) culture inbasal medium 6.2a with 2 g/L iron choline citrate and feed medium 6.2awith 0.56 g/l iron choline citrate; (filled pentagon) culture in basalmedium 6.2a with 1 g/L iron citrate and feed medium 6.2a with 0.56 g/liron choline citrate. ▪ culture in RPMI based basal medium 3.1 with 0.2g/l iron choline citrate and feed medium 2 with 0.25 g/l iron citrate, ●culture in RPMI based basal medium 3.1 with 2 g/L iron choline citrateand feed medium 2 with 0.25 g/l iron citrate; ♦ culture in RPMI basedbasal medium 3.1 with 1 g/L iron citrate and feed medium 2 with 0.25 g/liron citrate.

FIG. 10 : Two fed-batch cultures of CHO-K1 GS derived cell linesproducing Rituximab were cultivated in parallel in basal medium 6.2 GSand feed medium 6.2 GS, both with optimized AA ratios. Shown are (A)viable cell concentration [1×10⁶ cell/mL], (B) viability [%] of CHO-K1GS cells producing Rituximab and (C) final product concentration after a14 days cultivation process [mg/L].

DETAILED DESCRIPTION Definitions

The general embodiments “comprising” or “comprised” encompass the morespecific embodiment “consisting of”. Furthermore, singular and pluralforms are not used in a limiting way. Terms used in the course of thispresent invention have the following meaning.

The term “cell culture medium” as used herein is a medium to culturemammalian cells comprising a minimum of essential nutrients andcomponents such as vitamins, trace elements, salts, bulk salts, aminoacids, lipids, carbohydrates in a preferably buffered medium (preferablypH about 7.0, pH=7.3-6.6, pH=7.0). Non limiting examples for such cellculture media include commercially available media like RPMI, DMEM:F12,DMEM, Hams/F12 etc. as well as proprietary media from various sources(e.g. medium 6.2). The cell culture medium may be a basal cell culturemedium. The cell culture medium may also be a basal cell culture mediumto which the feed medium and/or additives have been added. The cellculture medium may also be referred to as fermentation broth, if thecells are cultured in a fermenter or bioreactor.

The term “basal medium” or “basal cell culture medium” as used herein isa cell culture medium to culture mammalian cells as defined below. Itrefers to the medium in which the cells are cultured from the start of acell culture run and is not used as an additive to another medium,although various components may be added to the medium. The basal mediumserves as the base to which optionally further additives or feed mediummay be added during cultivation, i.e., a cell culture run. The basalcell culture medium is provided from the beginning of a cell cultivationprocess. In general, the basal cell culture medium provides nutrientssuch as carbon sources, amino acids, vitamins, bulk salts (e.g. sodiumchloride or potassium chloride), various trace elements (e.g. manganesesulfate), pH buffer, lipids and glucose. Major bulk salts are usuallyprovided only in the basal medium and must not exceed a final osmolarityin the cell culture of about 280-350 mOsmo/kg, so that the cell cultureis able to grow and proliferate at a reasonable osmotic stress.

The term “feed” or “feed medium” as used herein relates to a concentrateof nutrients/a concentrated nutrient composition used as a feed in aculture of mammalian cells. It is provided as a “concentrated feedmedium” to avoid dilution of the cell culture, typically a feed mediumis provided at 10-50 ml/L/day, preferably at 15-45 ml/L/day, morepreferably at 20-40 ml/L/day and even more preferably at 30 ml/L/daybased on the culture starting volume (CSV, meaning the start volume onday 0) in the vessel. The feeding rate is to be understood as an averagefeeding rate over the feeding period. A feed medium typically has higherconcentrations of most, but not all, components of the basal cellculture medium. Generally, the feed medium substitutes nutrients thatare consumed during cell culture, such as amino acids and carbohydrates,while salts and buffers are of less importance and are commonly providedwith the basal medium. The feed medium is typically added to the (basal)cell culture medium/fermentation broth in fed-batch mode. However, thefeed may be added in different modes like continuous or bolus additionor via perfusion related techniques (chemostat or hybrid-perfusedsystem). Preferably, the feed medium is added daily, but may also beadded more frequently, such as twice daily or less frequently, such asevery second day. The addition of nutrients is commonly performed duringcultivation (i.e., after day 0). In contrast to the basal medium, thefeed consists of a highly concentrated nutrient solution (e.g. >6×) thatprovides all the components similar to the basal medium except for‘high-osmolarity-active compounds’ such as major bulk salts (e.g., NaCl,KCl, NaHCO₃, MgSO₄, Ca(NO₃)₂). Typically a >6×-fold concentrate orhigher of the basal medium without or with reduced bulk salts maintainsgood solubility of compounds and sufficiently low osmolarity (e.g.270-1500 mOsmo/kg, preferably 310-800 mOsmo/kg; medium 6.2 feedosmolarity is about 1500 mOsmo/kg due to high glucose, salts andoptimized AA) in order to maintain osmolarity in the cell culture atabout 270-550 mOsmo/kg, preferably at about 280-450 mOsmo/kg, morepreferably at about 280-350 mOsmo/kg.

The cell culture medium, both basal medium and/or feed medium may beserum-free, chemically defined or chemically defined and protein-free. A“serum-free medium” as used herein refers to a cell culture medium forin vitro cell culture, which does not contain serum from animal origin.This is preferred as serum may contain contaminants from said animal,such as viruses, and because serum is ill-defined and varies from batchto batch. The basal medium and the feed medium according to theinvention are serum-free.

A “chemically defined medium” as used herein refers to a cell culturemedium suitable for in vitro cell culture, in which all components areknown. More specifically it does not comprise any supplements such asanimal serum or plant, yeast or animal hydrolysates. It may comprisehydrolysates only if all components have been analysed and the exactcomposition thereof is known and can be reproducibly prepared. The basalmedium and the feed medium according to the invention are preferablychemically defined.

A “protein-free medium” as used herein refers to a cell culture mediumfor in vitro cell culture comprising no proteins, except for proteinsproduced by the cell to be cultured, wherein protein refers topolypeptides of any length, but excludes single amino acids, dipeptidesor tripeptides. Specifically growth factors such as insulin andinsulin-like growth factor (IGF) are not present in the medium.Preferably, the basal medium and feed medium according to the presentinvention are chemically defined and protein-free.

As used herein, the “medium platform”, or “media platform” consists of abasal cell culture medium, which is provided from the beginning of acell cultivation process and a feed medium, which is added to the basalcell culture medium during cultivation. Optionally further additives,such as glucose, may be added during the cell cultivation process. Thefeed medium may be supplied in any kind of fed batch process mode (e.g.continuous, with changing feed rates or as bolus feed additions).

The term “commercially available media/media systems” as used hereinrefers to commercially available cell culture media with completelyknown composition. These media serve as references for the media of thepresent invention due to the requirement for exact nutrient composition.Commercially available media are, e.g., DMEM:F12 (1:1), DMEM, HamsF12,and RPMI. The feed medium of the commercial media used herein wereprepared as a 12-fold concentrate of the basal medium without bulksalts. The term “commercially available media systems” relate to asystem comprising of a commercially available basal cell culture medium,such as DMEM:F12 (1:1), DMEM, HamsF12, and RPMI and a feed medium, whichis the respective concentrated basal medium (e.g., 12-fold concentrated)without or with reduced bulk salts.

As used herein, “lx” means the standard concentration normally used in aparticular basal medium, “2×” means twice the standard concentration,etc. The feed medium is for example preferably a 6× to 20× solution,i.e., 6 to 20-fold the standard concentration in the basal medium thatis used for amino acid optimization without considering bulk salts suchas sodium chloride or potassium chloride. However, the skilled personwill understand that the cell culture requirements are different during,e.g., the exponential growth phase and the protein production phase.Thus, preferably the basal medium and the feed medium are adapted tothese altered requirements. Hence, the amino acid ratios in the feedmedium are typically different to the amino acid ratios in the basalmedium.

The term “cell cultivation” or “cell culture” includes cell cultivationand fermentation processes in all scales (e.g. from micro titer platesto large-scale industrial bioreactors, i.e. from sub mL-scale to >10.000L scale), in all different process modes (e.g. batch, fed-batch,perfusion, continuous cultivation), in all process control modes (e.g.non-controlled, fully automated and controlled systems with control ofe.g. pH, temperature, oxygen content), in all kind of fermentationsystems (e.g. single-use systems, stainless steel systems, glass waresystems). In a preferred embodiment of the present invention the cellculture is a mammalian cell culture and is a batch or a fed-batchculture.

The term “fed-batch” as used herein relates to a cell culture in whichthe cells are fed continuously or periodically with a feed mediumcontaining nutrients. The feeding may start shortly after starting thecell culture on day 0 or more typically one, two or three days afterstarting the culture. Feeding may follow a preset schedule, such asevery day, every two days, every three days etc. Alternatively, theculture may be monitored for cell growth, nutrients or toxic by-productsand feeding may be adjusted accordingly. Common monitoring methods foranimal cell culture are described in the experimental part below. Ingeneral, the following parameters are often determined on a daily basisand cover the viable cell concentration, product concentration andseveral metabolites such as glucose or lactic acid (an acidic wastemetabolite that reduces the pH and is derived from cellular glucoseconversion), pH, osmolarity (a measure for salt content) and ammonium(growth inhibitor that negatively affects the growth rate and reducesviable biomass). Compared to batch cultures (cultures without feeding),higher product titers can be achieved in the fed-batch mode. Typically,a fed-batch culture is stopped at some point and the cells and/or theprotein of interest in the medium are harvested and optionally purified.

The terms “vitality” and “viability” are synonymously used and refers tothe % viable cells in a cell culture as determined by methods known inthe art, e.g., trypan blue exclusion with a Cedex device based on anautomated-microscopic cell count (Innovatis AG, Bielefeld). However,there exist of number of other methods for the determination of theviability such as fluorometric (such as based on propidium iodide),calorimetric or enzymatic methods that are used to reflect the energymetabolism of a living cell e.g. methods that use LDHlactate-dehydrogenase or certain tetrazolium salts such as alamar blue,MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) or TTC(tetrazolium chloride).

The term “amino acid” as used herein refers to the twenty natural aminoacids that are encoded by the universal genetic code, typically theL-form (i.e., L-alanine, L-arginine, L-asparagine, L-aspartic acid,L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine andL-valine). The amino acids (e.g., glutamine and/or tyrosine) may beprovided as dipeptides with increased stability and/or solubility,preferably containing an L-alanine (L-ala-x) or L-glycine extension(L-gly-x), such as glycyl-glutamine and alanyl-glutamine. Further,cysteine may also be provided as L-cystine. The term “amino acids” asused herein encompasses all different salts thereof, such as (withoutbeing limited thereto) L-arginine monohydrochloride, L-asparaginemonohydrate, L-cysteine hydrochloride monohydrate, L-cystinedihydrochloride, L-histidine monohydrochloride dihydrate, L-lysinemonohydrochloride and hydroxyl L-proline, L-tyrosine disodium dehydrate.The exact form of the amino acids is not of importance for thisinvention, unless characteristics such as solubility, osmolarity,stability, purity are impaired. Typically and preferably, L-arginine isused as L-arginine×HCl, L-asparagine is used as L-asparagine×H₂O,L-cysteine is used as L-cysteine×HCl×H₂O, L-cystine is used asL-cystine×2 HCl, L-histidine is used as L-histidine×HCl×H₂O andL-tyrosine is used as L-tyrosine×2 Na×2 H₂O, wherein each preferredamino acid form may be selected independent of the other or together orany combination thereof. Also encompassed are dipeptides comprising oneor two of the relevant amino acids. For example L-glutamine is oftenadded in the form of dipeptides, such as L-alanyl-L-glutamine to thecell culture medium for improved stability and reduced ammonium built upin storage or during long-term culture. This is also valid forL-glycine-containing dipeptides or other L-alanine-containingdipeptides, which are considered for calculation of the amino acidratios.

The term “all amino acids in the medium” or “total amino acid content”as use herein refers to the sum of the “amino acids” as defined above inmM. In a dipeptide, each amino acid counts separately, thus 1 mMalanyl-glutamine is counted as 1 mM L-alanine and 1 mM L-glutamine(molar ratio 1:1). Likewise in L-cystine each cysteine countsseparately, thus 1 mM L-cystine is counted as 2 mM L-cysteine (molarratio 1:2). Typically the total amino acid content is about 5 to20-fold, preferably about 7 to 15-fold and more preferably about 10-foldhigher in the concentrated feed medium compared to the basal cellculture medium. The total amino acid content of the basal mediumaccording to the invention may be about 25 to 150 mM, preferably about30 to 130 mM, more preferably about 35 to 120 mM and even morepreferably about 40 to 100 mM. The total amino acid content of the feedmedium may be about 100 to 1000 mM, preferably about 200 to 900 mM, morepreferably about 300 to 800 mM and even more preferably about 400 to 700mM. Other amino acids that are not directly coded by the universalgenetic code, such as L-ornithine, hydroxyl L-proline or metabolitesthereof such as taurine may further be present in the basal cell culturemedium or the feed medium, but these are not counted for the total aminoacid content.

The term “amino acid ratio” as used herein refers to the ratio of themolar concentration of each amino acid related to the molarconcentration of the reference amino acid. A molar ratio is calculatedfor every amino acid related to the reference amino acid (with the unit[mM/mM]). For the calculation of the amino acid ratios according to thepresent invention L-isoleucine is used as reference amino acid (althoughtheoretically other amino acids can be used as reference amino acidssuch as phenylalanine or methionine). This may further be referred to asmolar ratio relative to isoleucine (mM/mM). Typically, a reference aminoacid can be easily measured with statistically low standard variationsand is provided in similar concentration ranges in commonly used media.

The term “spent media amino acid ratio adjustment” means that aminoacids are adjusted only based on spent media analysis but withoutconsideration of cellular and metabolic demands and specificintracellular or extracellular rates. Thus, an amino acid analysis isperformed for samples taken from the cell culture supernatant on variousdays and amino acids below a certain threshold are to be supplemented inthe basal and feed medium.

The term “iron choline citrate” as used herein relates to the chemicalcompound ferric choline citrate falling under the CAS No. 1336-80-7 thatforms an iron choline citrate complex. Common synonyms used are e.g.ferrocholinate citrate, ferric choline citrate, choline citrate, iron(Ill) choline citrate, choline ferric citrate, tricholine citrate,choline ferric citrate, 2-Hydroxyethyl-trimethyl-ammonium,2-Hydroxypropane-1,2,3-tricarboxylate, boxylato(4-)ferrate(1-),ethanaminium,2-hydroxy-n,n,n-trimethyl-,hydroxy(2-hydroxy-1,2,3-propanetricar. Thiscompound may be added as an iron carrier to both the basal and the feedmedium. Preferably iron choline citrate with a molariron:choline:citrate ratio of 2:3:3 (ferric choline citrate, CAS-Number1336-80-7, molecular weight Mw=991.5 g/mol+/−49.57 g/mol due to 5%crystal water content, iron complex with iron content of about10.2-12.4%, molecule ratio for iron:choline:citrate of 2:3:3, moleculeformula C₃₃H₅₇Fe₂N₃O₂₄, which is e.g. obtainable from Dr. Paul LohmannGmbH KG) is used. However, other suitable iron choline citratestructures may be used at equimolar amounts based on the ironconcentration, e.g. iron:choline:citrate at a ratio of 1:1:1, molecularweight of Mw=348.11 g/mol or (iron):choline:citrate at a ratio of(2):3:3, molecular weight of Mw=501.61 g/mol, C₂₁H₄₇N₃O₁₀ (sum formulawithout iron). Compared to the state of the art iron sources used incommercially available cell culture media such as iron phosphate, ironpyrophosphate or iron citrate, the usage of iron choline citratecontributes to significantly higher product titers at equimolar amounts.

The terms “polypeptide” or “protein” or “product” or “product protein”or “amino acid residue sequence” are used interchangeably. These terms“refer to polymers of amino acids of any length. These terms alsoinclude proteins that are post-translationally modified throughreactions that include, but are not limited to glycosylation, glycation,acetylation, phosphorylation, oxidation, amidation or proteinprocessing. Modifications and changes, for example fusions to otherproteins, amino acid sequence substitutions, deletions or insertions,can be made in the structure of a polypeptide while the moleculemaintains its biological functional activity. For example certain aminoacid sequence substitutions can be made in a polypeptide or itsunderlying nucleic acid coding sequence and a protein can be obtainedwith similar or modified properties. Amino acid modifications can beprepared for example by performing site-specific mutagenesis orpolymerase chain reaction mediated mutagenesis on its underlying nucleicacid sequence. The terms “polypeptide”, “protein”, “product” and“product protein” thus also include, for example, fusion proteinsconsisting of an immunoglobulin component (e.g. the Fc component) and agrowth factor (e.g. an interleukin), antibodies or any antibody derivedmolecule formats or antibody fragments.

The term “protein of interest” or “product of interest” or “polypeptideof interest” includes proteins, polypeptides, fragments thereof,peptides, fusion proteins all of which can be expressed in the selectedhost cell. Typically, the protein of interest is a recombinant protein,i.e., a protein encoded by a recombinant DNA resulting from molecularcloning. Such proteins of interest can be antibodies, enzymes,cytokines, lymphokines, adhesion molecules, receptors and derivatives orfragments thereof, and any other polypeptides that can serve as agonistsor antagonists and/or have therapeutic or diagnostic use or can be usedas research reagent. Preferably the protein of interest is a secretedprotein or protein fragment, more preferably an antibody or antibodyfragment or an Fc-fusion protein. The “product of interest” may also bean antisense RNA, tRNA, rRNAs, other RNAs being part of riboproteins orother regulatory RNAs.

The term “gene of interest”, “desired sequence”, “polynucleotide ofinterest” or “desired gene” as used herein have the same meaning andrefer to a polynucleotide sequence of any length that encodes a productof interest. The gene may further comprise regulatory sequencespreceding (5′ non-coding or untranslated sequences) and following (3′non-coding or untranslated sequences) the coding sequence. The selectedsequence can be full length or a truncated gene, a fusion or taggedgene, and can be a cDNA, a genomic DNA, or a DNA fragment. It isgenerally understood that genomic DNA encoding for a polypeptide or RNAincludes non-coding regions (i.e. introns) that are spliced from maturemessenger RNA (mRNA) and are therefore not present in cDNA encoding forthe same polypeptide or RNA. It can be the native sequence, i.e.naturally occurring form(s), or can be mutated, or comprising sequencesderived from different sources or otherwise modified as desired. Thesemodifications include codon optimizations to optimize codon usage in theselected host cell or tagging. Furthermore they can include removal oradditions of cis-acting sites such as (cryptic) splice donor, acceptorsites and branch points, polyadenylation signals, TATA-boxes, chi-sites,ribosomal entry sites, repeat sequences, secondary structures (e.g. stemloops), binding sites for transcription factors or other regulatoryfactors, restriction enzyme sites etc. to give just a few, but notlimiting examples. The selected sequence can encode a secreted,cytoplasmic, nuclear, membrane bound or cell surface polypeptide.

Cell Culture Media and Amino Acid Ratios

The 20 standard amino acids that are encoded by the universal geneticcode (L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine and L-valine), play an essentialrole for protein synthesis since they provide the building blocks forboth cellular proteins and for the protein of interest (e.g. monoclonalantibodies). Thus, amino acids interact manifold in the cellularmetabolism. They are taken up from the cell culture medium in specificamounts, they are inter-converted within cellular metabolism, eitherdirected into host cell proteins or into the product protein, excretedby a cell as by-product, and are connected at various points to thecellular metabolic catabolism and anabolism, e.g., between amino acidmetabolism and citric acid cycle. In both basal and feed medium optimalcomposition, concentrations, and ratios of amino acids need to beprovided for optimal nutritional supply throughout the life cycle(seeding, lag phase, exponential growth phase, transition phase,stationary phase, death phase characterized by significant decrease incell viability) of a cell cultivation. However, the ratio of amino acidsseems to be more important than the actual exact concentration of eachindividual amino acid.

Thus, in one aspect of the present invention a basal cell culture mediumfor culturing mammalian cells is provided comprising the following aminoacids at a molar ratio relative to isoleucine (mM/mM) of:L-leucine/L-isoleucine of about 1.2-2.2; L-phenylalanine/L-isoleucine ofabout 0.5-0.9, L-tyrosine/L-isoleucine of about 1.5-2.7,L-threonine/I-isoleucine of about 1.0-1.9, and L-valine/L-isoleucine ofabout 1.0-1.9, wherein the basal cell culture medium has a total aminoacid content of about 25 to 150 mM amino acids. In one embodiment themolar ratio relative to isoleucine (mM/mM) is: L-leucine/L-isoleucine ofabout 1.2-2.1, preferably about 1.3-1.8, more preferably about 1.5-1.8and even more preferably about 1.7; L-phenylalanine/L-isoleucine ofabout 0.5-0-9, preferably about 0.6-0.9, more preferably about 0.6-0.8and even more preferably about 0.7; L-tyrosine/L-isoleucine of about1.6-2.6, preferably about 1.7-2.5, more preferably about 1.9-2.3 andeven more preferably about 2.1; L-threonine/I-isoleucine of about1.1-1.8, preferably about 1.2-1.8, more preferably about 1.3-1.6 andeven more preferably about 1.5; and L-valine/L-isoleucine of about1.1-1.9, preferably about 1.2-1-8, more preferably about 1.3-1.6 andeven more preferably about 1.5. In certain embodiments the medium of thepresent invention further comprises L-lysine at a molar ratio relativeto isoleucine of about 1.6-2.9, preferably of about 1.7-2.8, morepreferably of about 1.8-2.7, more preferably of about 2.0 to 2.5 andeven more preferably of about 2.2. In certain embodiments the basalmedium of the present invention further comprises L-tryptophan at amolar ratio relative to isoleucine of about 0.3-0.5, preferably of about0.3-0.5, more preferably of about 0.3-0.4, more preferably of about0.3-0.4 and even more preferably of about 0.4; or L-proline at a molarratio relative to isoleucine of about 1.6-3.0, preferably of about1.7-2.8, more preferably of about 1.8-2.7, more preferably of about2.0-2.5 and even more preferably of about 2.3, or L-methionine at amolar ratio relative to isoleucine of about 0.4-0.7, preferably of about0.4-0.6, more preferably of about 0.4-0.6, more preferably of about0.5-0.6 and even more preferably of about 0.5. In certain embodimentsthe molar ratios of L-tryptophan, L-proline and L-methionine relative toL-isoleucine are as defined above. The total amino acid content in thebasal cell culture medium may be about 25-150 mM, preferably about30-130 mM, more preferably about 35-120 mM, and even more preferablyabout 40-100 mM.

Preferably the amino acid ratios for L-leucine, L-phenylalanine,L-threonine, L-valine and L-tyrosine and optionally further forL-lysine, L-tryptophane, L-proline and/or L-methionine relative toL-isoleucine are within 30%, 25%, 20% or 10% of the ratios provided forbasal medium 6.2 in table 2a.

More specific exemplary amino acid ratios of the basal cell culturemedium (basal medium 6.2) of the present invention are provided in tableA below in direct comparison to amino acid ratio in selected commercialbasal cell culture media.

TABLE A Amino acid ratios for each amino acid with reference isoleucine(Ile) for basal cell culture medium. Amino acid ratio (concentration AA/concentration reference AA isoleucine [mM/mM] Amino Medium acid (AA)DMEM_F12 DMEM HamsF12 RPMI 6.2 L-Alanine 0.1 3.3 — — L-Arginine 1.7 0.533.3 2.5 2.1 L-Asparagine 0.1 — 3.3 0.9 1.8 L-Aspartic 0.1 — 3.3 0.4 1.3Acid L-Cysteine 0.7 0.5 6.7 1.9 1.6 L-Glutamic 0.1 — 3.3 0.4 0.9 AcidL-Glutamine 14.1 7.3 194 55.7 46.4 L-Glycine 0.6 0.5 3.3 30.0 24.7L-Histidine 0.4 0.3 3.3 0.3 0.9 L-Isoleucine 1.0 1.0 1.0 1.0 1.0L-Leucine 1.1 1.0 3.3 1.0 1.7 L-Lysine 1.2 1.0 6.75 0.6 2.2 L-Methionine0.3 0.3 1.0 0.3 0.5 L-Phenyl- 0.5 0.5 1.0 0.2 0.7 alanine L-Proline 0.4— 10.0 0.5 2.3 L-Serine 0.6 0.5 3.3 0.8 2.1 L-Threonine 1.1 1.0 3.3 0.41.5 L-Tryptophan 0.1 0.1 0.3 0.1 0.5 L-Tyrosine 0.5 0.5 1.0 0.3 2.1L-Valine 1.1 1.0 3.3 0.5 1.5

In another aspect of the invention a feed medium for culturing mammaliancells is provided comprising the following amino acids at a molar ratiorelative to isoleucine (mM/mM) of: L-leucine/L-isoleucine of about2.3-4.2, L-phenylalanine/L-isoleucine of about 0.6-1.1,L-threonine/I-isoleucine of about 1.3-2.4, and L-valine/L-isoleucine ofabout 1.1-2.0, wherein the feed medium has a total amino acid content ofabout 100 to 1000 mM. In one embodiment the molar ratio relative toisoleucine (mM/mM) is: L-leucine/L-isoleucine of about 2.4-4.0,preferably of about 2.6-3.9, more preferably of about 2.9-3.5 and evenmore preferably of about 3.2; L-phenylalanine/L-isoleucine of about0.6-1.1, preferably of about 0.7-1.0, more preferably of about 0.8-0.9and even more preferably of about 0.9; L-threonine/I-isoleucine of about1.4-2.3, more preferably of about 1.5-2.2, more preferably of about1.7-2.0 and even more preferably of about 1.8; and L-valine/L-isoleucineof about 1.2-2.0, preferably of about 1.3-1.9, more preferably of about1.4-1.7 and more preferably of about 1.6.

In one embodiment the feed medium further comprises L-tyrosine at amolar ratio relative to isoleucine of about 0.6-1-1 and/or L-lysine at amolar ratio relative to isoleucine of about 1.1-2.1. Preferably tyrosineis present in the feed medium at a ratio of about 0.6-1.0, preferably ofabout 0.7-1.0, more preferably of about 0.7-0.9 and even more preferablyof about 0.8. Preferably lysine is present in the feed medium at a ratioof about 1.2-2.0, preferably of about 1.3-1.9, more preferably of about1.4-1.8 and even more preferably of about 1.6. Preferably the molarratio of L-tyrosine and L-lysine are as defined above. In certainembodiments the feed medium of the present invention further comprisesL-tryptophan at a molar ratio relative to isoleucine of about 0.3-0.6,preferably of about 0.3-0.6, more preferably of about 0.4-0.5, morepreferably of about 0.4-0.5 and even more preferably of about 0.5; orL-proline at a molar ratio relative to isoleucine of about 0.9-1.8,preferably of about 1.0-1.7, more preferably of about 1.1-1.6, morepreferably of about 1.2-1.5 and even more preferably of about 1.4, orL-methionine at a molar ratio relative to isoleucine of about 0.4-0.8,preferably of about 0.4-0.7, more preferably of about 0.5-0.7, morepreferably of about 0.5-0.6 and even more preferably of about 0.6. Incertain embodiments the molar ratios of L-tryptophan, L-proline andL-methionine relative to L-isoleucine are as defined above. The totalamino acid content in the basal cell culture medium may be about100-1000 mM, preferably about 200 to about 900, more preferably about300 to about 800, and even more preferably about 400 to about 700 mM.

Preferably the amino acid ratios for L-leucine, L-phenylalanine,L-threonine and L-valine, and optionally further for L-tyrosine,L-lysine, L-tryptophane, L-proline and/or L-methionine relative toL-isoleucine are within 30%, 25%, 20% or 10% of the ratios provided forfeed medium 6.2 in table 6.

More specific exemplary amino acid ratios of the feed medium (feedmedium 6.2) of the present invention are provided in the table B belowin direct comparison to amino acid ratio in selected commercial feedmedia.

TABLE B Amino acid ratios for each compound with reference isoleucine(Ile) for feed medium. Amino acid ratio (concentration AA/ concentrationreference AA isoleucine [(mM)/(mM)]) Amino acid (AA) Feed DMEM_F12 FeedRPMI Feed medium 6.2 L-Alanine 0.1 — — L-Arginine 1.7 2.5 1.0L-Asparagine 0.1 0.9 3.2 L-Aspartic Acid 0.1 0.4 0.2 L-Cysteine 3.7 0.30.7 L-Glutamic Acid 0.1 0.4 0.3 L-Glutamine 11.0 — — L-Glycine 0.6 0.81.1 L-Histidine 0.4 0.3 0.6 L-Isoleucine 1.0 1.0 1.0 L-Leucine 1.1 1.03.2 L-Lysine 1.2 0.6 1.6 L-Methionine 0.3 0.3 0.6 L-Phenylalanine 0.50.2 0.9 L-Proline 0.4 0.5 1.4 L-Serine 0.6 0.8 3.2 L-Threonine 1.1 0.41.8 L-Tryptophan 0.1 0.1 0.5 L-Tyrosine 0.3 0.4 0.8 L-Valine 1.1 0.5 1.6

The feed medium is added as a concentrated feed medium to the basal cellculture medium or the culture medium. For example, the feed medium maybe added at about 10-50 ml/L/day, preferably at about 15 to 45 ml/L/day,more preferably at about 20-40 ml/L/day and even more preferably atabout 30 ml/L/day based on the culture starting volume (CSV). The rate(volume/day) for addition of the feed medium to the cell culture inml/L/day (volume in ml added per liter of culture starting volume in thevessel per day) is to be understood as an average rate over the feedingperiod and the added volume may vary between individual additions duringthe feeding period. Also feeding may be stopped about 1 to 3 days priorto termination of the culture and/or harvest. It is preferable to add asmall volume to avoid dilution of other nutrients in the cell cultureand to maintain the culture volume as constant as possible. The feedmedium may be added continuously, several times a day, daily or everysecond day. Preferably, said feed medium is added starting on day 0, day1 or day 2 every day or every second day.

The basal cell culture medium and/or the feed medium of the inventionare serum-free and preferably chemically defined or chemically definedand protein-free. Further the basal cell culture medium and the feedmedium of the invention are suitable for culturing mammalian cells,i.e., they are a basal mammalian cell culture medium and a mammalianfeed medium, respectively. The basal cell culture medium and the feedmedium of the invention is suitable for culturing all kinds of mammaliancells, such as rodent or human cells, wherein rodent cells arepreferred. More preferably the mammalian cell is a Chinese hamster ovarycell (CHO), such as a CHO-K1 cell, a CHO-DG44 cell, a DuxB11 cell or aCHO GS deficient cell, most preferably the cell is a CHO-DG44 cell or aCHO GS deficient cell.

The basal cell culture medium comprising the amino acid ratio of theinvention may further comprise the iron choline citrate at aconcentration as described for the basal cell culture medium hereinbelow. Similarly, the feed medium comprising the amino acid ratio of theinvention may further comprise the iron choline citrate at aconcentration as described for the feed medium herein below.

Culture Media and Iron Carrier

In mammalian cell culture, iron is required as a trace element. In vivo,iron is bound primarily by ferritin and transferrin in serum. A typicaliron source in cell culture media is transferrin. In advanced serum-freeor even protein-free mammalian cell culture media, several aspectsrelated to iron need to be solved, such as the identification of asuitable iron carrier, the poor bioavailability of iron, theidentification of adequate physiological concentration ranges (withminimal/no negative effects on e.g. cell viability due the presence ofharmful free radicals in vitro with respect to the underlying toxicityof ferric compounds), the complex binding behavior (iron can bind to aplurality of substances within a medium formulation and thereby caneasily become biologically unavailable for the cell culture), oxidationstatus, and optimal cell culture performance (e.g. titer).

In the present invention the chemical compound iron choline citrate isprovided as a novel iron-carrier in mammalian cell culture with improvedcharacteristics compared to established iron-carriers used in cellcultivation.

Thus, in another aspect the invention provides a basal cell culturemedium for culturing mammalian cells comprising iron choline citrate ata concentration of about 0.1 to 5.0 mM, about 0.2 to 2.0 mM, about 0.2to 1.0 mM or about 0.4 to 1.0 mM.

In yet another aspect, the invention provides a feed medium comprisingiron choline citrate at a concentration of about 0.4 to 5.0 mM, about0.4 to 1.0 mM, or about 0.5 to 1 mM, preferably about 0.5 to 0.6 mM.

The concentrations of iron choline citrate in the basal medium and thefeed medium are based on iron choline citrate with a molariron:choline:citrate ratio of 2:3:3 (ferric choline citrate, CAS-Number1336-80-7, molecular weight Mw=991.5 g/mol+/−49.57 g/mol due to 5%crystal water content, iron complex with iron content of about10.2-12.4%, molecule ratio for iron:choline:citrate of 2:3:3, moleculeformula C₃₃H₅₇Fe₂N₃O₂₄). However, other iron choline citrate structuresare encompassed by the invention and may be used at equimolar amountsbased on the iron concentration, e.g. iron:choline:citrate at a ratio of1:1:1, molecular weight of Mw=348.11 g/mol. This means for example that1 mM iron choline citrate with a molar iron:choline:citrate ratio of2:3:3 equates to 2 mM iron choline citrate with a molariron:choline:citrate ratio of 1:1:1.

The use of iron choline citrate as iron carrier results in increasedproduct titers. Additionally, compared to other iron sources that areestablished as iron carriers such as iron citrate, the novel ironcarrier iron choline citrate is typically chemically characterized by ahigher purity compared to iron citrate. The higher potential lot-to-lotvariability of established iron carriers such as iron citrate can causenegative effects in manufacturing of biopharmaceuticals (e.g.reproducibility in manufacturing is negatively affected). Iron cholinecitrate can be used in both basal and/or feed medium, preferably ironcholine citrate is added to both the basal medium and the feed medium.Compared to other iron sources used in cell culture media such as iron(III) phosphate or iron (III) pyrophosphate, the use of iron cholinecitrate leads to an improved culture performance, e.g., significantlyhigher product titers. The basal cell culture medium comprising ironcholine citrate according to the invention may further comprise thenovel amino acid ratio of the basal medium according to the invention,as described above. Similarly, the feed medium comprising the ironcholine citrate according to the invention may further comprise thenovel amino acid ratio of the feed medium according to the invention asdescribed above.

The basal cell culture medium and/or the feed medium of the inventionare serum-free, preferably chemically defined or chemically defined andprotein-free. Further the basal cell culture medium and the feed mediumof the invention are suitable for culturing mammalian cells, i.e., theyare a basal mammalian cell culture medium and a mammalian feed medium,respectively. The basal cell culture medium and the feed medium of theinvention is suitable for culturing all kinds of mammalian cells, suchas rodent or human cells, wherein rodent cells are preferred. Morepreferably the mammalian cell is a Chinese hamster ovary cell (CHO),such as a CHO-K1 cell, a CHO-DG44 cell, a DuxB11 cell or a CHO GSdeficient cell, most preferably the cell is a CHO-DG44 cell or a CHO GSdeficient cell.

Culture Media and Other Components

A cell culture medium to culture mammalian cells may further compriseessential nutrients and components such as vitamins, trace elements,salts, bulk salts, lipids or lipid precursors and carbohydrates in apreferably buffered medium. Also growth factors may be added to thebasal cell culture medium or the feed medium, e.g., recombinantinsulin-like growth factor (IGF) or recombinant insulin.

Non-limiting examples for suitable vitamins are biotin (B7), calciumpantothenate, cyanocobalamin (B12), folic acid, myoinositol, niacinamid(B3), pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin (B2)and/or thiamine (B1). Non-limiting examples for trace elements areammonium molybdate, ammonium vanadate, cupric sulfate, nickel sulfate,sodium selenite, sodium silicate, and zinc sulfate and/or zinc chloride.Non-limiting examples of lipid precursors are choline chloride,ethanolamine, glycerol, inositol, linoleic acid, fatty acids,phospholipids or cholesterol-related compounds.

Further, salts may be, without being limited thereto, calcium chloride,calcium nitrate, magnesium chloride, magnesium sulfate, potassiumchloride and/or sodium chloride. One function of the salt is to adjustthe osmolarity in the medium. Preferably the osmolarity of a basal cellculture medium does not go beyond an optimal range of typically between280-350 mOsmo/kg. Typically the osmolarity of a concentrated feed mediumis <2000 mOsmo/kg, preferably <1500 mOsmo/kg, more preferably <1000mOsmo/kg. The osmolarity of the feed medium may be higher, but shouldnot increase the osmolarity in the cell culture upon addition beyond theoptimal range of 270-550 mOsmo/kg, preferably of 280-450 mOsmo/kg, morepreferably of 280-350 mOsmo/kg.

Preferably, the feed medium of the present invention in any of itsembodiments has reduced or low salt content. A reduced or low saltcontent means, e.g., a total salt concentration of about 100 mM or less,preferably of about 50 mM or less (e.g. a feed medium without sodiumchloride, and a reduced concentration of potassium chloride). A reducedlow salt content in the feed medium of the present invention isespecially preferred when the feed medium is combined with the basalcell culture medium of the present invention for use as regular growthmedium.

The most important contributors to osmolarity are sodium ions, chlorideions, and bicarbonate as well as glucose and other carbon sources e.g.amino acids. For the medium developer it is a challenge to create a highconcentrated nutrient mixture and a powder formulation for manufacturingthat meets the following requirements: preferably a x-fold concentrateof basal medium composition (positive impact for supply chain managementand regulatory aspects), provide essential nutrients and nutrients thatcannot be synthesized by the cell itself in adequate amounts (preferablyin as rational-balanced composition), overcome solubility aspects forfeed concentrates, remove bulk salts due to osmolarity reasons, avoidtoxic ranges, design a powder formulation that requires an carboncarrier for galenic reasons. Furthermore, for a common fed-batch processthe feed medium needs to be concentrated to minimize the culture volumeover the cultivation period. The size of the bioreactor may actuallycause feeding constrains that allow only total feed dosages ofapproximately 30% (25-35%) of the culture starting volume.

Carbohydrates may be, but are not limited to glucose, mannose,galactose, fructose, sucrose or glucosamine etc. These carbohydrates canbe added directly to the basal cell culture medium and/or the feedmedium or may be added separately to the cell culture. Other energysources include, but are not limited to sodium pyruvate.

Mammalian cells should be cultured at a neutral pH, such as from aboutpH 6.5 to about pH 7.5, preferably from about pH 6.6 to about pH 7.3,more preferred at a pH of about 7. Hence buffering agents should beadded to the basal cell culture medium. For the feed medium the pH maybe slightly outside said range, as long as the addition of the feedmedium does not bring the pH of the cell culture outside this range,since the feed medium is added as a concentrate. Preferred ranges forthe pH in a feed medium are from about 6 to about 8. Suitable bufferingagents include, but are not limiting to Hepes, phosphate buffers (e.g.,potassium phosphate monobasic and potassium phosphate dibasic and/orsodium phosphate dibase anhydrate and sodium phosphate monobase), phenolred, sodium bicarbonate and/or sodium hydrogen carbonate.

Generally, the feed medium comprises nutrients that are consumed duringcell culture, such as amino acids and carbohydrates, while salts andbuffers are of less importance. Some salts may therefore be omittedentirely from a feed medium.

Cell Culture Performance

The basal cell culture medium and/or the feed medium of the presentinvention or the cell culture medium platform comprising the chemicallydefined basal medium and the chemically defined feed medium of theinvention result in improved cell culture performance. The term“improved cell culture performance” as used herein comprises, e.g.,significantly improved product titers, improved cell growth (e.g. viablecell counts, cell viability), and favorable phenotypic behavior of acell culture process such as reduced overflow metabolism of unwanted andtoxic by-products (e.g. reduced lactate formation). It also contributesto reduced osmolarity levels in a cell cultivation process.

The basal cell culture medium and/or the feed medium of the presentinvention meet the cell specific requirements and metabolic needs of amammalian cell culture during the time course of cell cultivation. Inother words it meets (i) the cell specific needs of a mammalian cell,(ii) in a cell cultivation system, (iii) throughout the lifecycle of acultivation run (which is about 10-20 days). Mammalian cells in culturehave different nutritional requirements in different phases of a cellculture process. Yet, ideally, only one optimal basal medium and onlyone (or quite few) optimal feed medium/media need to be designed toenable the design of robust, safe, and efficient bioprocesses. The basalmedium and/or feed medium provided herein fulfill this need.

The basal cell culture medium and/or the feed medium of the presentinvention have improved cell culture performance. Non limiting examplesfor improved cell culture performance are increase of product titers,improved viable cell concentrations and/or cell viabilities. Also thecell expansion may be improved, which is needed for the inoculationtrain in a scale-up procedure. For example, cultivation scales arestepwise increased from thaw of a cell bank (mL scale) to the productionscale (>10.000 L scale). The better the growth in each N-x stage is(with N-stage meaning the final production scale and N-x meaning thecell expansion stages before final production stage usually in batchmode), the faster and the better each transfer to the next stage canoccur. Specifically, better cell growth and higher viable cellconcentrations allow that N-x cultivations can be performed with reducedrun times (hence faster). Better cell growth and higher viable cellconcentrations also result in improved transfers resulting in an overallimproved performance. For example, when a certain N-x stage should beinoculated with a certain seeding cell density and the viable cellconcentration is high, a relatively low volume of cell culture needs tobe transferred from one stage to the next (transfer of inoculum volumeper CSV is defined as spit ratio, usually 1:5 to 1:20 is common). Thismeans that at the same time only a reduced volume of “used” cell culturemedium is transferred from one stage to the next and a maximal volume of“new” media can be added to the next stage (constant overall cultivationvolumes). This also results in improved overall cell culture performancein the final N-stage (e.g. increased product titer). With the novelbasal cell culture medium and feed medium provided in the presentinvention all of these stages are improved. The positive effects of thenovel iron carrier iron choline citrate and/or the novel amino acidratios are not limited to basal medium and feed medium in the finalproduction stage. It is also shown that the positive effects of themedia platform apply to the N-x stages, in particular for the amino acidratios. These positive effects are also maintained in the case of mediamodifications in the N-x stages. For example, typically MTX(methotrexate) is provided in early stages of the inoculation train inorder to maintain the selection pressure in mammalian cell culture usingrecombinant cell lines such as CHO cell lines, preferably CHO-DG44 celllines. Also in such examples, the application of the basal cell culturemedium and/or feed medium or the media platform of the invention resultsin significantly improved viable cell concentrations.

Cell Culture/Addition of Feed Medium

The addition of a nutrient concentrate named “feed medium” is requiredfor the standard fed-batch application in contrast to the common batchfermentation, where no concentrated feed medium is added to the cultureduring the entire cultivation. In contrast to a batch application, it iswell known that the cell culture performance e.g. maximal viable cellcount, final product titer, metabolic waste accumulation issignificantly improved in a fed-batch process due to the replenishmentof nutrients, vitamins, salts and other components. Typically themaximal amount of feed solution added to the culture during thecultivation time depends on technical, but also on metabolism-drivenaspects: the maximal volume of the bioreactor constrains the total feedvolume to be added, whereas a non-technical feed dosage is applied tomeet the real cellular nutrient demand at any time during thecultivation. Furthermore, dependent on the cell line and process mode,the feed addition can be added continuously e.g. in small scale 2-80 L(development, less work intense) with a constant feed rate of, e.g.,5-60 ml feed/L/d or with a non-continuous (large scale manufacturing,more work intense) approach e.g. in 2000-10.000 L large scale tominimize the risk of contamination. Typical intervals for feed additionsduring an e.g. 11-day fed-batch cultivation can vary between severaltimes a day, daily or every 2-4 days, and often depend on the actualnutrient level, growth phase, ● culture conditions such as pH or thenutrient demand of the culture.

Lactate/Carbon Dioxide/Glucose

In most cell cultures a non-ideal nutrient combustion for major carboncan be determined due to an overflow-metabolism. This means, that themajor carbon source glucose is utilized ineffectively and by thiscontributes to an increase of organic acids e.g. lactic acid. Theincreased level of lactic acid can contribute to a pH drop below 6.65and this would negatively affect the buffer capacity of the culturemedium and thus the culture viability. For such reason, the CO2concentration in the culture atmosphere is reduced at the beginning ofthe exponential growth phase in order to minimize the acid level in theculture medium.

Cell Lines and Cell Culture

The basal cell culture medium and/or the feed medium or the mediumplatform of the present invention can be applied to all mammalian celllines. However, the media of the present invention may further besuitable for other eukaryotic cells, such as yeast, plant or insectcells. The mammalian cell according to the invention may be oocytes,embryonic stem cells, hematopoietic stem cells or any type ofdifferentiated cells. Preferably, the mammalian cell is a human, simian,murine, goat, bovine, sheep, pig cell or rodent cell line such as rat,rabbit or hamster. The mammalian cell may be an isolated primary cell ora cell line. Preferred cell lines or “host cells” for the production ofrecombinant biopharmaceuticals are human, monkey, or rodent cell lines(mice, rat or hamster). Preferred human cells are PER.C6 or HEK 293cells.

More preferred are rodent cells, such as hamster cells, preferablyBHK21, BHK TK-, Chinese Hamster ovary cells (CHO), CHO-K1, CHO-DXB11(also referred to as CHO-DUKX or DuxB11), CHO-DUKX B1, CHO-S, CHO-DG44and CHO glutamine synthetase (GS) deficient cells or thederivatives/progenies of any of such cell lines. Particularly preferredare CHO-DG44, CHO-DUKX, CHO-K1, CHO-S, CHO-DG44 GS deficient cell linesand BHK21, and even more preferred CHO-DG44 cells, CHO GS deficientcells (such as a CHO-K1 GS deficient cell) and CHO-DUKX cells.Furthermore, murine myeloma cells, preferably NS0 and Sp2/0 cells or thederivatives/progenies of any of such cell lines are also known asproduction cell lines for biopharmaceutical proteins.

All cells and cell lines may be used in all kind of cell cultivations,e.g., ranging from plastic microtiter plates (nL to mL scale) toindustrial scale stainless steel bioreactors (L to kL scale), they alsoinclude any type of disposable system and all kinds of process controlstrategies from non-controlled systems to fully controlled systemscomprising e.g. advanced online monitoring and advanced controlstrategies. Suitable culture conditions for mammalian cells are known inthe art. Mammalian cells may be for example cultured in suspension orattached to a solid surface.

Non-limiting examples of mammalian cells, which can be used with themedia of the present invention are summarized in Table C.

TABLE C Suitable exemplary mammalian production cell lines CELL LINEREFERENCE NUMBER NS0 ECACC No. 85110503 Sp2/0-Ag14 ATCC CRL-1581 BHK21ATCC CCL-10 BHK TK⁻ ECACC No. 85011423 HaK ATCC CCL-15 2254-62.2 (BHK-21derivative) ATCC CRL-8544 CHO ECACC No. 8505302 CHO wild type ECACC00102307 CHO-DUKX (=CHO duk⁻, ATCC CRL-9096 CHO/dhfr⁻) CHO-DUKX B11 ATCCCRL-9010 CHO-DG44 Urlaub et al., Cell 33 (2), 405-412, 1983; LifeTechnologies A1097101 CHO Pro-5 ATCC CRL-1781 CHO-S Life TechnologiesA1136401; CHO-S is derived from CHO variant Tobey et al. 1962 Lec13Stanley P. et al, Ann. Rev. Genetics 18, 525-552, 1984 V79 ATCC CCC-93HEK293 ATCC CRL-1573 COS-7 ATCC CRL-1651 HuNS1 ATCC CRL-8644 Per.C6Fallaux, F. J. et al, Human Gene Therapy 9 (13), 1909-1917, 1998 CHO-K1ATCC CCL-61, ECACC 85051005 CHO-K1/SF ECACC 93061607 CHO-K1 GS glutaminesynthetase (GS) deficient cells derived from CHO-K1 CHOZN GS GSdeficient cells derived from CHO-K1 (SAFC ECACC 85051005)

Said production cells are cultivated preferentially under conditionsthat allow the cells to proliferate. Furthermore, said production cellsare cultivated preferentially under conditions, which are favorable forthe expression of the desired gene(s) and/or the protein of interest.The protein of interest is than isolated from the cells and/or the cellculture supernatant. Preferably the protein of interest is recoveredfrom the culture medium as a secreted polypeptide, or it can berecovered from host cell lysates if expressed without a secretorysignal.

Typically, it is necessary to purify the protein of interest from otherrecombinant proteins, host cell proteins and contaminants in a way thatsubstantially homogenous preparations of the protein of interest areobtained. As a first step cells and/or particulate cell debris may beremoved from the culture medium or lysate. Typically, the product ofinterest is then purified from contaminant soluble proteins,polypeptides and nucleic acids, for example, by fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC, Sephadex chromatography, chromatography on silica or on acation exchange resin such as DEAE.

Use of the Culture Medium

The basal cell culture medium or the feed medium of the invention can beused as a growth medium, as an inoculum medium, as medium for cellexpansion or for cell line development, including transfection,amplification or both. Further, the basal cell culture medium or thefeed medium may be used, preferably in combination, for producing aprotein of interest from a mammalian cell expressing said protein ofinterest.

Specifically the basal medium and the feed medium of the invention maybe used in a method of culturing a mammalian cell comprising thefollowing steps: a) providing mammalian cells, b) culturing the cells inthe basal cell culture medium of the invention, and c) optionally addingthe feed medium of the invention to the basal cell culture medium;wherein the cells are cultured under conditions that allow the cells toproliferate. Preferably the feed medium is also used in said method.

The basal medium and the feed medium of the invention may further beused in a method of producing a protein of interest comprising thefollowing steps: a) providing mammalian cells comprising a gene ofinterest encoding the protein of interest, b) culturing the cells in thebasal cell culture medium of the invention, and c) optionally adding thefeed medium of the invention to the basal cell culture medium, and d)optionally separating and/or isolating and/or purifying said protein ofinterest from the cell culture; wherein the cells are cultured underconditions that allow expression of the protein of interest. Preferablythe feed medium is used in said method. The feed medium may be added tothe cells cultured in the basal cell culture medium at about 10-50ml/L/day, preferably at about 15-45 ml/L/day, more preferably at about20-40 ml/L/day and more preferably at about 30 ml/L/day based on theculture starting volume (CSV), wherein the medium may be addedcontinuously or as a bolus several times a day, two times a day, onceper day, every second day or every third day. Preferably the feed mediumis added starting on day 0, 1, 2 or 3. The rate (volume/day) foraddition of the feed medium to the cell culture in ml/L/day (feed volumein ml added per liter of culture starting volume in the vessel per day)is to be understood as an average rate over the feeding period and theadded volume may vary between individual additions during the feedingperiod. Also, feeding may be stopped about 1 to 3 days prior totermination of the culture and/or harvest.

Separating the protein of interest from the cell culture can be done bye.g., centrifugation, filtration or any other method known in the artfor separating the supernatant comprising the protein from cells or celldebris. This may include lysis if the protein is producedintracellularly. Purification of the protein of interest from the cellculture means isolating one or a few proteins from a complex mixture,such as a cell culture supernatant or a lysate by methods known in theart, such as precipitation, chromatography or gel electrophoresis.

The basal medium and the feed medium of the invention may be used in alarge-scale cell culture, preferably a cell culture of 100 L or more,more preferably of 1000 L or more or even more preferably of 10000 L ormore.

The protein of interest may be an antibody, an enzyme, a cytokine, alymphokine, an adhesion molecule, a receptor, or derivatives orfragments thereof, and any other polypeptide that can serve as agonistor antagonist and/or have therapeutic or diagnostic use or can be usedas research reagent. The protein of interest may be for example anantibody, such as Rituximab, or an Fc-fusion protein. Preferably theantibody is a monoclonal IgG1 antibody with a heavy and light chainhaving the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2 or with aheavy and light chain having the amino acid sequence of SEQ ID NO: 3 andSEQ ID NO:4. The Fc-fusion protein preferably has the amino acidsequence of SEQ ID NO: 5.

Proteins of interest may also be proteins/polypeptides, which are usedto change the properties of host cells within the scope of so-called“Cell Engineering”, such as e.g. anti-apoptotic proteins, chaperones,metabolic enzymes, glycosylation enzymes and the derivatives orfragments thereof, but are not restricted thereto.

Especially, desired proteins/polypeptides or proteins of interest arewithout being limited thereto, e.g., insulin, insulin-like growth factor(IGF1), hGH, tPA, cytokines, such as interleukines (IL), e.g. IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFNbeta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF), suchas TNF alpha and TNF beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF,MCP-1, VEGF and nanobodies. Also included is the production oferythropoietin or any other hormone growth factors and any otherpolypeptides that can serve as agonists or antagonists and/or havetherapeutic or diagnostic use. The method according to the invention canalso be advantageously used for production of antibodies, such asmonoclonal, polyclonal, multispecific and single chain antibodies, orfragments derived thereof, e.g. Fab, Fab′, F(ab′)2, Fc andFc′-fragments, heavy and light immunoglobulin chains and their constant,variable or hypervariable region as well as Fv- and Fd-fragments.

The term “antibody”, “antibodies”, or “immunoglobulin(s)” as used hereinrelates to proteins selected from among the globulins, which are formedas a reaction of the host organism to a foreign substance (=antigen)from differentiated B-lymphocytes (plasma cells). They serve to defendspecifically against these foreign substances. There are various classesof immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably theantibody is an IgG antibody, more preferably an IgG1 antibody. The termsimmunoglobulin and antibody are used interchangeably. Antibody includesa polyclonal, monoclonal, monospecific, bi-specific, multi-specific, asingle chain antibody, an antigen-binding fragment of an antibody (e.g.,an Fab or F(ab′)2 fragment), a disulfide-linked Fv, etc. Antibodies canbe of any species and include chimeric and humanized antibodies.“Chimeric” antibodies are molecules in which antibody domains or regionsare derived from different species. For example the variable region ofheavy and light chain can be derived from rat or mouse antibody and theconstant regions from a human antibody. In “humanized” antibodies onlyminimal sequences are derived from a non-human species. Often only theCDR amino acid residues of a human antibody are replaced with the CDRamino acid residues of a non-human species such as mouse, rat, rabbit orllama. Sometimes a few key framework amino acid residues with impact onantigen binding specificity and affinity are also replaced by non-humanamino acid residues. Antibodies may be produced through chemicalsynthesis, via recombinant or transgenic means, via cell (e.g.,hybridoma) culture, or by other means.

Immunoglobulins are tetrameric polypeptides composed of two pairs of aheterodimer each formed by a heavy and light chain. Stabilization ofboth the heterodimers as well as the tetrameric polypeptide structureoccurs via interchain disulfide bridges. Each chain is composed ofstructural domains called “immunoglobulin domains” or “immunoglobulinregions” whereby the terms “domain” or “region” are usedinterchangeably. Each domain contains about 70-110 amino acids and formsa compact three-dimensional structure. Both heavy and light chaincontain at their N-terminal end a “variable domain” or “variable region”with less conserved sequences which is responsible for antigenrecognition and binding. The variable region of the light chain is alsoreferred to as “VL” and the variable region of the heavy chain as “VH”.

The term “Fab fragment(s) “(Fragment antigen-binding=Fab) or “Fab”consist of the variable regions of both antibody heavy and light chains(VH and VL) which are held together by the adjacent constant regions(CH1 and CL). These may be formed by protease digestion, e.g. withpapain, from conventional antibodies, but similar Fab fragments may alsobe produced in the meantime by genetic engineering. Further antibodyfragments include “F(ab′)2 fragments” or “F(ab′)2”, which may beprepared by proteolytic cleaving with pepsin or by genetic engineeringin which both Fab arms of an antibody are still linked via inter-heavychain disulfide bridges located within the hinge region.

The immunoglobulin fragments composed of the CH2 and CH3 domains of theantibody heavy chain are called “Fc fragments”, “Fc region” or “Fc”because of their crystallization propensity (Fc=fragmentcrystallizable). These may be formed by protease digestion, e.g. withpapain or pepsin from conventional antibodies but may also be producedby genetic engineering. The N-terminal part of the Fc fragment mightvary depending on how many amino acids of the hinge region are stillpresent.

The term “Fc-fusion protein” describes polypeptides which contain as afusion partner a natural or modified (e.g. substitutions, deletions,insertions) Fc region of an immunoglobulin. Fc fusion proteins can beeither naturally occurring proteins (e.g. antibodies) or engineeredrecombinant proteins (e.g. TNF receptor-Fc fusion protein or a VH regionfused to an Fc region). The Fc-fusion proteins can exist either asmonomers or as multimers whereby polypeptides can have identical ordifferent sequences, might contain linker sequences between the twofusion partners and/or part of the hinge region or modified hingeregions or the polypeptide is fused directly to the CH2 domain.

Using genetic engineering methods it is possible to produce shortenedantibody fragments which consist only of the variable regions of theheavy (VH) and of the light chain (VL). These are referred to as “Fvfragments” (Fragment variable=fragment of the variable part) or “Fv”.Since these Fv-fragments lack the covalent bonding of the two chains bythe cysteines of the constant chains, the Fv fragments are oftenstabilized. It is advantageous to link the variable regions of the heavyand of the light chain by a short peptide fragment, e.g. of 10 to 30amino acids, preferably 15 amino acids. In this way a single peptidestrand is obtained consisting of VH and VL, linked by a peptide linker.An antibody protein of this kind is known as a “single-chain-Fv” or“scFv”. Examples of scFv-antibody proteins of this kind are known fromthe prior art. In addition, more than one VH and/or VL region can belinked together.

In recent years, various strategies have been developed for preparingscFv as a multimeric derivative. This is intended to lead, inparticular, to recombinant antibodies with improved pharmacokinetic andbiodistribution properties as well as with increased binding avidity. Inorder to achieve multimerisation of the scFv, scFv were prepared asfusion proteins with multimerisation domains. The multimerisationdomains may be, e.g. the CH3 region of an IgG or coiled coil structure(helix structures) such as Leucine-zipper domains. However, there arealso strategies in which the interaction between the VH/VL regions ofthe scFv is used for the multimerisation (e.g. dia-, tri- andpentabodies). By diabody the skilled person means a bivalent homodimericscFv derivative. The shortening of the linker in a scFv molecule to 5-10amino acids leads to the formation of homodimers in which an inter-chainVH/VL-superimposition takes place. Diabodies may additionally bestabilized by the incorporation of disulphide bridges. Examples ofdiabody-antibody proteins are known from the prior art.

By minibody the skilled person means a bivalent, homodimeric scFvderivative. It consists of a fusion protein which contains the CH3region of an immunoglobulin, preferably IgG, most preferably IgG1 as thedimerisation region which is connected to the scFv via a Hinge region(e.g. also from IgG1) and a linker region. Examples of minibody-antibodyproteins are known from the prior art.

By triabody the skilled person means a: trivalent homotrimeric scFvderivative. ScFv derivatives wherein VH-VL is fused directly without alinker sequence lead to the formation of trimers.

The skilled person will also be familiar with so-called miniantibodieswhich have a bi-, tri- or tetravalent structure and are derived fromscFv. The multimerisation is carried out by di-, tri- or tetramericcoiled coil structures. In a preferred embodiment of the presentinvention, the gene of interest is encoded for any of those desiredpolypeptides mentioned above, preferably for a monoclonal antibody, aderivative or fragment thereof.

The term “antibody derived molecule(s)” is used interchangeably with“antibody derived fragments” or “antibody fragments” and refers topolpypeptides which contain only part(s) of one or more antibodydomain(s) or region(s) and/or complete domain(s) or region(s). Theantibody fragments can be either a) forming a molecule on their own, b)linked with each other in different combinations, c) fused tonon-antibody sequences, d) fused or linked to non-polypeptide (e.g.radionucleotides) or d) any combination of the above. These polypeptidescan exist either as monomers or as multimers whereby polypeptides canhave identical or different sequences.

EXAMPLES

Materials and Methods

Cell Line

CHO cell lines (CHO-DG44) were adapted to serum-free media conditionsand further transfected with DNA to produce recombinant products such asmonoclonal antibodies, fusion proteins or bi/multi-specific proteinsthat are relevant for industrial manufacturing. Specifically, twoproprietary BI HEX (Boehringer-Ingelheim High Expression) CHO-DG44derived CHO cell lines that were independently adapted to serum-freemedia (named HEX I and HEX II) expressing different IgG constructs wereused. These cells are DHFR⁻ (dihydrofolate-reductase) deficient andmethotrexate is used as selection marker. If not otherwise stated thecells used in the experiments are CHO-DG44 (HEX II) cells expressingRituximab as recombinant protein with a heavy chain having the aminoacid sequence of SEQ ID NO: 1 and a light chain having the sequence ofSEQ ID NO: 2, which is secreted into the culture medium. This cell lineis referred to as CHO2, CHO-DG44 Rituximab or CHO2 (CHO-DG44) Rituximabin the following.

Analytical Methods

Cell concentrations and cell viabilities were determined by the trypanblue exclusion method using a CEDEX (Type 5.00, version 2.2) automatedcell analyzer (Roche Innovatis, Bielefeld, Germany). The concentrationsof produced recombinant proteins in the medium, such as IgG antibodieswere quantified by a Konelab 60i (Thermo Scientific, Dreieich, Germany)analyzer based on photometrical methods or by the use of a HPLC method.The Konelab 60i instrument was also used for the quantification ofmetabolites such as glucose, lactic acid (lactate), glutamine,glutamate, and ammonium in the cell culture supernatants. Amino acidconcentrations were determined by use of a GC 6890N/FID gaschromatograph (Agilent Technologies GmbH & Co.KG, Waldbronn, Germany).Amino acid analysis was performed by the EZ-faast protocol fromPhenomenex (Aschaffenburg, Germany). Osmolarity profiles were analyzedby an osmomat auto device (Gonotec GmbH, Berlin, Germany). This methodis based on the cryoscopic freezing point of a particular solution,which is proportional to the amount of dissolved particles. Dissolvedcarbon dioxide pCO₂, dissolved oxygen pO₂ and pH were determined on adaily basis with a Rapidlab 248/348 instrument (Siemens HealthcareDiagnostics GmbH, Eschborn, Germany). These instruments and the requiredmethods are well known in the art and used for process monitoring andcontrol in biopharmaceutical process development and manufacturing.

Shake Flask Cultivation

Shake flask (Corning B.V. Life Sciences, Amsterdam, Netherlands)experiments were generally performed in small scale, with a workingvolume in the range of 60-500 ml in batch mode (no feed addition duringcultivation) or in fed-batch mode (with a standard feed rate of 30ml/L/d nutrient feed addition during cultivation). Viable cellconcentration at inoculation was typically set to 0.3×10⁶ cells/ml inevery experiment. The shake flasks cultivations were derived from thesame inoculum pre-culture (thawing, expansion of cells in a seed train,with respect to cell age) to ensure comparability between differentexperimental settings if required. For cell cultivation a standard shakeflask incubator (Infors AG, Bottmingen, Switzerland) was used at ashaking rate of 120 rpm, a temperature set point of 37° C., and humiditywas set to 70%. The analytical methods as described above were used tomeasure the standard process parameter on a daily basis, which are totaland viable cell count, cell viability, metabolite concentrations andother relevant cell culture parameters such as dissolved oxygen pO₂(DO), dissolved carbon dioxide pCO₂ (DCO) content or pH. This was doneroutinely throughout the cultivation to monitor and control thecultivation conditions for each experimental setup. In fed batchexperiments, a concentrated feed solution was added in fed-batchexperiments as a bolus addition of 1.8 ml feed per day to a culturestarting volume of 60 ml (corresponding to 30 ml/L/d nutrient feed ratebased on the culture starting volume), starting on day 2, in anuncontrolled shake flasks system.

Batch and Fed-Batch Mode

For the production of recombinant proteins and antibodies, typicallyfed-batch processes are used in the final production stage, while batchcultivations are mainly performed in the cell expansion stages prior tothe final production stage. A series of batch cultures is referred to asseed train during cell expansion, meaning that cells are transferred ineach expansion step into cultivations vessels with larger cultivationvolumes. Batch processes in the final production stage do generally notresult in high productivity and are therefore rarely used formanufacturing recombinant proteins. In fed-batch processes concentratedfeed medium is added during cultivation to compensate for replenishmentof nutrients with fresh medium. These processes achieve a higherproductivity and are therefore used predominantly in recombinant proteinproduction. In contrast to the batch mode, a replenishment of nutrientsby adding concentrated feed medium also reduces inhibition of cellgrowth by unwanted metabolic by-products such as lactate or ammonium.Typically fed-batch processes are started at a volume much lower thanthe maximal capacity of a stirred tank so that concentrated nutrientsolutions can be added over the bioreactor cultivation time.

Bioreactor Cultivation

The bioreactor experiments were performed in a controlled 2-L system(Boehringer-Ingelheim proprietary multi-fermenter system) with a startvolume of 1.8 L or in a controlled 48-mini-bioreactor system with astarting volume of maximal 15 ml. The fully controlled bioreactors wereperformed in batch or fed-batch mode. In fed-batch a concentratedfeeding solution was continuously added by a feed pump from cultivationusually from day 1-3 onwards with a feeding rate of 30 ml/L/d (based onthe culture starting volume). The seeding density was set to 0.3×10⁶cells/ml similar to the shake flasks system. The expansion of cells overa longer time frame followed a standard seed train protocol for cellgrowth and culture splits in order to ensure phenotypic stability. Thisprocedure ensures comparability between different experimental settingsat different time points. For a typical bioreactor cultivation, astandard process format consists of a pH range from 7.10-6.95 (+/−0.25)including a pH shift from day 3, a DO set point of 30-60% (airsaturation), a constant stirring rate of 140 rpm (4-blade rushtonturbine stirrer), and a temperature set point of 36.5-37° C. Theanalytical methods as described above were used to determine the majorculture parameters such as cell count, cell viability, and major carbonmetabolite concentrations to provide an ideal nutrient supply to thecell culture. In contrast to the shake flask experiments, in thebioreactor systems pH and pO₂ is monitored online. The offline processparameters and set-points were fully controlled by a control software(Siemens, Munich Germany) using an automatic closed-loop system formonitoring, e.g., the pH control, nutrient feed addition, temperaturecontrol, stirring and gassing.

Example 1

CHO2 (CHO-DG44) Rituximab cells were cultured in a RPMI based basalmedium with RPMI amino acid (AA) ratios versus optimized amino acid (AA)ratios with different total cumulative amounts of amino acids. Themedium composition for medium 4 (medium 4.0, 4.1, 4.2 and 4.3) havingRPMI AA ratios and medium (medium 5.0, 5.1 and 5.2) having optimized AAratios with total cumulative amounts of amino acids ranging from 22mM-66 mM (37 mM only in medium 4) are shown in Table 1 and thecorresponding amino acid ratios in Table 2.

Minor variations in total AA concentrations are due to variations inmolecular weight and minimal variation of used amino acid powders. Theaim of this experiment was to demonstrate the impact of optimized aminoacid ratios at different total cumulative amino acid levels. Theexperiment was performed in batch mode in duplicates (N=2).

TABLE 1 Compositions of Media 4.0, 4.1, 4.2 and 4.3 and Media 5.0, 5.1and 5.2 Basal medium 4 Basal medium 5 Unit WFI 0.800 0.800 l/l AApremixed powder (RPMI AA 3.08 (37 mM; medium 4.0) g/l ratios)* 3.73 (45mM; medium 4.1) 5.58 (67 mM; medium 4.2) 1.86 (23 mM; medium 4.3) AApremix powder (optimized AA 4.23 (44 mM; medium 5.0) g/l ratios)** 6.34(66 mM; medium 5.1) 2.11 (22 mM; medium 5.2) Powder GM RPMI 86638 (table1b) 4.37 4.37 g/l NaHCO₃ 3.0 3.0 g/l Monoethanolamin stock sol. 800 800μl/l (12.22 g/l stock solution) Sigma Aldrich Chemie Iron cholinecitrate (991.5 g/mol); 0.2 0.2 g/l Dr. Paul Lohmann GmbH KG Selenic acid(25.79 mg/l stock sol.) 100.0 100.0 μl/l Putrescine × 2HCl [mg/l] 4.84.8 mg/l Insulin (5 g/l stock sol.) 2 2 ml/l Chemical defined lipids(Gibco 5.0 5.0 ml/l Life Technol. 92_0239DK) Hepes 3.57 3.57 g/l NaCl6.00 6.00 g/l MgSO₄ 0.049 0.049 g/l KCl 0.40 0.40 g/l Ca(NO₃)₂ *4H₂O0.10 0.10 g/l Glucose 1.50 1.50 g/l Pluronic 1.00 1.00 g/l 40% NaOHadjust pH to 7.1 adjust pH to 7.1 ml/l Water for injection (WFI) add 1.0add 1.0 l/l Total glucose 5.00 5.00 g/l *Gln, Ile and Cys were addedseparately using a stock solution **Gln and Ile were added separatelyusing a stock solution

TABLE 1a Amino Acid Ratios for Medium 4 (non- optimized) and Medium 5(optimized) Medium 4 (RPMI AA Medium 5 (optimized AA Amino Acid molarratios) molar ratios) L-Alanine — — L-Arginine 2.5 2.1 L-Asparagine 0.91.8 L-Aspartic Acid 0.4 1.3 L-Cysteine 1.9 1.6 L-Glutamic Acid 0.4 0.9L-Glutamine — 46.4 L-Glycine 0.8 24.7 L-Histidine 0.3 0.9 L-Isoleucine1.0 1.0 L-Leucine 1.0 1.7 L-Lysine 0.6 2.2 L-Methionine 0.3 0.5L-Phenylalanine 0.2 0.7 L-Proline 0.5 2.3 L-Serine 0.8 2.1 L-Threonine0.4 1.5 L-Tryptophan 0.1 0.4 L-Tyrosine 0.4 2.1 L-Valine 0.5 1.5

TABLE 1b Composition Powder GM RPMI 86638 COMPONENT [g/L] Sodiumphosphate dibasic (anhyd.) 0.8 Choline chloride 0.003 i-Inositol 0.035L-Glutathione reduced 0.001 Biotin 0.0002 Cyanocobalamin (Vitamin B12)0.000005 D-Calcium pantothenate 0.00025 Folic Acid 0.001 Niacinamide0.001 Para-aminobenzoic acid 0.001 Pyridoxine × HCl 0.001 Riboflavin0.0002 Thiamine × HCl 0.001 D-Glucose 3.5 Ethanolamine × HCl 0.01563Putrescine × 2HCl 0.0048 Sodium selenite 0.000003458 Sum g/L 4.37

Materials and Methods:

The RPMI basal medium used in this experiment is based on thecommercially available RPMI medium R8755 (Mediatech catalog no. 90022PBor Sigma Aldrich catalog no. R8755) that was originally developed atRoswell Park Memorial Institute in 1966 by Moore and his co-workers(SAFC, Biosciences product information). For serum-free use it has beensupplemented as shown in table 1 containing sodium chloride (NaCl 6.0g/L), potassium chloride (KCl 0.4 g/L), magnesium sulfate (MgSO₄ 0.0488g/L) at a cumulative sum of bulk salts of 108.4 mmol/L.

The batch experiment was performed in 500 ml shake flasks with astarting volume of 125 ml. CHO2 (CHO-DG44) Rituximab cells were seededat 0.3×10⁶ cells/ml in medium 4, 4.1, 4.2 or 4.3 (RPMI AA ratios) andmedium 5, 5.1 or 5.2 (optimized AA ratios). The shake flasks cultureswere incubated at 36.5° C. in an incubator with 5% CO2 at day 0-3 and 3%CO2 from day 4 until the end of the cultivation.

The amino acid cysteine was provided in the powder formulation of medium5, but was added separately from a stock solution in medium 4. Formonitoring and control of the cultures, total cells, viable cells,viability, product concentration, glucose concentration, lactic acidconcentration, ammonium concentration and osmolarity were measured up today 7.

Results:

FIG. 1 (A-D) shows the results for cells cultured in RPMI medium withRPMI ratios (filled square) and optimized AA ratios (filled circles)i.e. viable cells concentration, viability, product concentration andlactate concentration at a total AA concentration of 44 mM. Highestviable growth and product concentration was achieved in cultures withoptimized AA ratios at different cumulative AA concentrations of 44 mM(FIGS. 1A and C) and 66 mM (FIGS. 1E and G). For example, the productconcentration was about 2.3-fold higher in cell cultures with optimizedAA ratios (FIG. 1C) at days 5 and 7, with a maximal productconcentration of 166 mg/L compared to a maximal product concentration of72 mg/L for cells grown in medium containing RPMI ratios. This wasaccompanied by a higher number of viable cells (FIG. 1A, up to 2.82×10⁶c/ml in medium 5 and 1.13×10⁶ c/ml in medium 4.1). Viability profile forboth cultures was in good agreement to each other and showed a cleardecrease from day 3 onwards from 98% down to 25% viability on day 7(FIG. 1B). The glucose concentration, ammonium concentration andosmolarity showed a similar tendency in both cultures. For example,glucose concentration was maintained greater than 1.0 g/L over thecultivation period for all cultures to avoid any limitation, and pH wasmaintained in typical ranges for cell culture process. This demonstratesthat all cultures were provided in sufficient amounts with major carbonsources such as glucose for cell growth, metabolism and productformation. As expected, the profile of the metabolic waste productlactate showed a growth dependent pattern, i.e. increased cellconcentrations contribute to higher amounts of the metabolic wasteproduct lactate (FIG. 1D). It should be noted that the lactic acidproduction is not always growth-associated and can be further understoodas an indicator for efficient glucose utilization (e.g. FIG. 4J).

Similar results were obtained at a total amino acid concentration of 66mM (FIG. 1 E-H) for cells cultured in medium containing RPMI ratios(filled square) and optimized AA ratios (filled circles). The productconcentration was about 3-fold higher in cell cultures with optimized AAratios (Figure G) at days 5 and 7, with a maximal product concentrationof 311 mg/L compared to a maximal product concentration of 96 mg/L forcells grown in medium containing RPMI AA ratios. This was accompanied byan increased number of viable cells (Figure E, up to 3.44×10⁶ c/ml inmedium 5.1 and 1.32×10⁶ c/ml in medium 4.2). Viability profile for bothcultures show a similar pattern, but cultures in medium 5.1 show aprolonged viability by approximately 1 day on day 5 (94% vs. 70% medium4.2). Viability of both cultures show a clear decrease from day 3onwards from 98% down to 55% and 25% viability on day 7 (FIG. 1F). Forlactate concentration, glucose concentration, ammonium concentration andosmolarity as well as for the pH progress a similar trend was observedas described above for cultures with a total amino acid concentration of44 mM. Overall viable cell concentration and product concentration washigher at a higher total amino acid concentration (compare 66 mM (FIGS.1E and G) versus 44 mM (FIGS. 1A and C)), particularly for cellscultured with optimized AA ratios (product concentration for mediumcontaining RPMI ratios: medium 4.2 (66 mM) vs. medium 4.1 (44 mM), day5: 96-72 mg/l=+24 mg/l; product concentration for medium containingoptimized AA ratios: medium 5.1 (66 mM) vs. medium 5.0 (44 mM) vs., day5: 290-166 mg/l=+123 mg/l).

A similar trend could also be observed for 22 and 36 mM total amino acidconcentration (FIG. 11 ). The maximal product concentration ofapproximately 45 mg/l (22 mM, medium 4.3, RPMI AA ratios; filledtriangle right) increased to a maximal product concentration of 65 mg/l(36 mM, medium 4.0, RPMI AA ratios; filled cross) with increasing totalamino acid concentration. However, this effect is smaller than theeffect associated with the optimized amino acid ratio if one compares amaximal product concentration of 117 mg/l (22 mM, medium 5.2, andoptimized AA ratios; filled square) vs. 45 mg/l (22 mM, medium 4.3, RPMIratios) as shown in FIG. 1I.

As can be taken from FIG. 1J, optimized AA ratios at the lowest testedtotal amino acid concentration of 22 mM (filled square) resulted inhigher productivity (maximal product concentration 117 mg/l, optimizedAA ratios, 22 mM) than RPMI AA ratios at the highest tested total aminoacid concentration of 66 mM (filled circles, maximal product conc. 96mg/ml, RPMI AA ratios). Thus, the highest productivity was achievedusing media with optimized AA ratios, with a maximal productconcentration of 117 mg/L (22 mM, optimized AA ratios; FIG. 1I, J), 166mg/l (44 mM, optimized AA ratios, FIG. 1C) and 290 mg/l (66 mM,optimized AA ratios, FIG. 1E). This shows that optimizing the AA ratiosstrongly increases productivity and that this can only be compensated toa very small extend by simply increasing the total AA concentration.

Example 2

Based on the optimized amino acid ratios in the basal medium 5 (RPMIbased), several amino acids were varied as a single component-approachin their molar concentration by +/−20% and +/−40% (calculation is basedon molar percentage for optimized AA ratios). Then, the performance wascompared to the control cultures grown in medium 5.3 (identical tomedium 5.0, but all amino acids were added individually as stocksolutions). All required amino acids were provided by concentrated stocksolutions to design a different medium composition. Thus, the media hada comparable total cumulative amino acid amount of approximately 43-44mM, but different amino acid ratios. In one experiment the variation ofsingle amino acid concentrations (single component-approach forL-arginine, L-asparagine, L-aspartate, L-histidine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine) by +20% and −20% vs. control medium5.3 was tested. A similar approach was performed for variations ofsingle amino acid concentrations (L-arginine, L-asparagine, L-aspartate,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine) by +40% and −40% compared to control. Theexperiment was performed in batch mode with a RPMI based medium.Variation of AA by +/−20% or +/−40% is indicated as (20) or (40) for thespecific medium used.

Materials and Methods:

This experiment was performed in 250 ml shake flasks with a startingvolume of 75 ml and 100 ml. In all cultures CHO2 (CHO-DG44) Rituximabcells were seeded at 0.3×10⁶ cells/ml in the control medium 5.3 (N=3)and the modified medium 5.3.1(20) (N=2) (single amino acid concentrationvaried by +/−20%), and medium 5.3.1(40) (single amino acid concentrationvaried by +/−40%). The shake flasks were incubated at 36.5° C. in anincubator (5% CO2 atmosphere was provided from day 0 to 3 followed by 3%CO2 until the end of the cultivation). Glucose was fed on day 2 and onday 4 and also on demand to keep the final glucose concentration between2.5 g/l and 4.5 g/l. L-glutamine was also added on demand.

The medium 5.3 (identical to medium 5.0, but all amino acids were addedindividually as stock solutions) served as the basis for thisexperiment. In total, 14 amino acids were tested for the +/−20% singlecomponent-approach: L-arginine, L-asparagine, L-aspartate, L-histidine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine and L-valine. In total 7 aminoacids were not tested: L-alanine, L-cysteine/L-cystine, L-glutamine,L-glutamate, L-glutamine, L-isoleucine and L-glycine. In total 15 aminoacids were tested for the +/−40% single component-approach: L-arginine,L-asparagine, L-aspartate, L-histidine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine and L-isoleucine. In total 6 aminoacids were not tested: L-alanine, L-cysteine/L-cystine, L-glutamine,L-glutamate, L-glutamine and L-glycine.

The amino acids not tested were qualified as metabolic waste that areproduced in excess in a cell culture (L-alanine, L-glycine,L-glutamate); are considered to be chemically instable due to oxidationof the compound or were not considered as being an essential compoundfor enhanced growth, especially after a cell peak e.g. L-glutamine.

Medium 5.3 was dissolved in water to form a 1.2-fold concentrate withoutamino acids to prepare Medium 5.3.1(20) for which all amino acids wereadded separately from stock solutions and adjusted with water. Medium5.3.1(40) was prepared as a 1.25-fold concentrate from Medium 5.3 forwhich all amino acids were added separately from stock solutions andadjusted with water.

Results:

The variation of single amino acid ratios showed that a reduction ofL-leucine, L-phenylalanine, L-threonine, L-valine or L-isoleucine in themedium with optimized AA ratios by 40% resulted in reduced productivity(FIGS. 2J and N). A small reduction in productivity was also observedwhen L-phenylalanine, L-valine or L-leucine was reduced by 20% (FIGS. 2Cand E-G).

For example, as shown in FIG. 2J, the average product concentrationranges from 129-184 mg/L on day 5 for the control medium 5.3 (optimizedAA ratios) and the modified medium 5.3.1(40) (single amino acid reducedby −40%) and from 127-186 mg/L on day 7. This wide range in final titerillustrates that the final product concentration is similar in mostmedia, but reduced in 5 cultures compared to the maximal control titerof 180 mg/L on day 7 (FIGS. 2J and 2N). The maximal productconcentration of 180 mg/L in the control medium 5.3 (optimized AAratios) is 180 mg/L, which is reduced in the modified media 5.3.1(40),with L-phenylalanine, L-valine, L-leucine, L-threonine or L-isoleucinereduced by −40%, to a maximal product concentration in the range of129-149 mg/L on culture day 5. A similar trend can be seen on day 7 witha product concentration of 179 mg/L in the control medium 5.3 and of127-143 mg/L in the five modified media.

Reducing L-leucine, L-phenylalanine, L-threonine, L-valine orL-isoleucine in the medium with optimized AA ratios by 40% furtherresulted in reduced viable cell concentrations, accompanied by adecrease in viability and an increase in waste metabolite (lactate)production following day 3 (FIG. 2H, 2L, 2M, 2O). No difference inviable cell concentration, cell viability or lactate production wasobserved when these amino acids were reduced by only 20% (see FIG. 2A,2B, 2D). A decrease of the viable cell concentration and viabilityoccurred almost in all cultures as expected due to the common nutrientdepletion and lack of feed addition in batch mode (FIGS. 2A, 2B, 2H, 2I,2L and 2M). Glucose was maintained above critical levels and lactateproduction followed a growth-associated kinetic as expected in allcultures (FIGS. 2K and O). Further no effect was observed on any of theother parameters measured such as osmolartiy, pCO₂ and pH.

The results show that reducing some of the amino acids negativelyinfluences the viable cell concentration and/or product formation.

No effect on productivity, viable cell concentration, cell viability orlactate concentration was observed in medium with amino acids increasedby 20 or 40% (data not shown).

Example 3

Based on the optimized amino acid ratios and the amino acids identifiedin example 2 in basal medium 5 (RPMI based), additional amino acids werevaried as a single component-approach in their molar concentration by−40% in a different medium background. This medium containing theoptimized AA ratios is further optimized for serum-free recombinantprotein production and is chemically defined and is superior to themodified RPMI medium used in the previous experiments. In thisexperiment, single amino acids were reduced in a batch mode todemonstrate the effect of optimized amino acid ratios in basal mediumunder controlled bioreactor conditions for pH, dissolved oxygen (DO) andtemperature.

Based on the optimized amino acid ratios in the basal medium 6.2, singleamino acids L-lysine, L-methionine, L-proline, L-tryptophan orL-tyrosine were reduced in their molar concentration by 40%, orL-tyrosine and L-lysine were both reduced by 20% or 40%. The resultingperformance was compared against the control culture medium containingthe optimized AA ratios. Compared were cells cultivated in controlmedium 6.2 (optimized AA ratios, AA added as premixed powder) andcontrol medium 6.4.1 (optimized AA ratios, AA added individually fromstock solutions) with cells cultivated in medium 6.4.9-medium 6.4.15(modified AA ratio, AA added individually from stock solutions). Alltested medium compositions had a comparable total cumulative amino acidconcentration of approximately 44 mM.

Materials and Methods:

Compared were cells cultured in control medium 6.2 (optimized AA ratios,AA added as premixed powder) and control medium 6.4.1 (optimized AAratios, AA added individually from stock solutions) with cells culturedin medium 6.4.9 (L-lysine and L-tyrosine −20%), medium 6.4.10 (L-lysineand L-tyrosine −40%), medium 6.4.11 (L-tyrosine −40%), medium 6.4.12(L-lysine −40%), medium 6.4.13 (L-methionine −40%), medium 6.4.14(L-tryptophan −40%), medium 6.4.15 (L-proline −40%). All tested mediumcompositions had a comparable total cumulative amino acid concentrationof 44-45 mM. All required amino acids were provided by concentratedstock solutions and added to medium 6.4.0 (identical to medium 6.2 and6.4.1, but without amino acids) to prepare the different mediumcompositions 6.4.1 and 6.4.9-15.

The experiment was performed in a 48-miniaturized bioreactor system witha starting volume of 14 ml. In all cell cultures CHO2 (CHO-DG44)Rituximab cells were seeded in the respective medium at 0.3×10⁶cells/ml. The bioreactors were incubated at 36.5° C. for the entirecultivation period and dissolved CO₂ was controlled between 2-15% toprevent toxic concentrations based on the pH set-point of(7.20-6.80)+/−0.2. Control cultures and experimental runs were performedin duplicates (N=2).

TABLE 2 Composition of Basal Medium (6.2, 6.3 and 6.4.0 without AA)Medium Medium Medium Components 6.2 6.3 6.4.0 Unit Total AA 44 mM 45 mM0 mM WFI 0.8 0.8 0.6 l/l AA premixed powder (optimized AA ratios)* 5.74— g/l AA premixed powder (RPMI ratios)* 4.83 — g/l Medium 6 powderwithout AA 10.10 10.10 10.10 g/l NaHCO3 4.5 4.5 4.5 g/l Iron cholinecitrate (ICC; MW = 991.5 g/mol) 0.2 0.2 0.2 g/l Dr. Paul Lohmann GmbH KGL-Ornithine × HCL 7.653 7.653 7.653 mg/l Putrescine × 2HCl [mg/l] 5.2375.237 5.237 mg/l Insulin (5 g/L stock sol.) (pharma Biocon)** 2 2 2 ml/lGlucose 5.00 5.00 5.00 g/l Succinic acid 1.50 1.50 1.50 g/l Taurine0.0011495 g/l L-Hydroxy-proline 0.0011248 g/l 40% NaOH on demand ondemand on demand ml/l WFI add 1.0 add 1.0 add 0.8 l/l *includes taurineand L-hydroxy-proline **insulin may be substituted with insulin-likegrowth factor (IGF) at a final concentration of 50 μl/l

TABLE 2a Amino Acid Ratios for Basal Medium 6.2 (optimized AA, AApremixed powder), 6.3 (non-optimized AA), 6.4.0.1 (optimized AA,control, AAs added separately) Amino Acids (AA) Medium 6.2 Medium 6.3Medium 6.4.0.1 Total AA 44 mM 45 mM 44 mM L-Alanine — — — L-Arginine2.13 2.5 2.1 L-Asparagine 1.82 0.9 1.8 L-Aspartic acid 1.31 0.4 1.3L-Cysteine 1.57 1.9 1.6 L-Glutamic acid 0.89 0.4 0.9 L-Glutamine 46.4046.4 46.40 L-Glycine 24.70 24.7 24.7 L-Histidine 0.91 0.3 0.9L-Isoleucine 1.00 1.0 1.0 L-Leucine 1.66 1.0 1.7 L-Lysine 2.24 0.6 2.2L-Methionine 0.51 0.3 0.5 L-Phenylalanine 0.72 0.3 0.7 L-Proline 2.270.5 2.3 L-Serine 2.08 0.8 2.1 L-Threonine 1.46 0.4 1.5 L-Tryptophan 0.370.1 0.4 L-Tyrosine 2.09 0.3 2.1 L-Valine 1.49 0.5 1.5

Results:

In the control culture (optimized AA ratios, medium 6.4.0.1) a maximalproduct concentration of 317 mg/L was measured on day 8. In all testcultures the maximal product concentration was reduced compared to thecontrol culture, ranging from 249 to 279 mg/L on day 8. Specifically,reducing L-lysine or L-tyrosine in the medium resulted in a productconcentration of 268 mg/L and 279 mg/L on day 8, respectively.Interestingly, reducing both L-lysine and L-tyrosine by 40% resulted inan even lower product concentration of 249 mg/L, indicating an additiveor even synergistic effect (FIG. 3C, filled cross). ReducingL-methionine, L-proline or L-tryptophan in the medium likewise resultedin a reduced product concentration on day 8 (265 mg/L, 274 mg/L, 277mg/L, respectively). In summary, the results show that the medium withthe optimized amino acid ratios resulted in the best productivity out ofthe tested media.

The growth profiles showed comparable results for the test media and forthe control media (FIGS. 3A and 3B). The maximal viable cellconcentration was a few days earlier in some of the cultures compared tothe control culture (filled squares) and even slightly higher. Forexample, the viable cell concentration for all cultures ranged from apeak cell density of 3.7 10⁶ cells/ml on day 4 to a lower cell densityof 2.8 10⁶ cells/ml on day 6 (control). Viability profiles for allcultures show a similar tendency with a sharp decrease on culture day 6.However, viability was even slightly higher in some of the test culturestowards the end of the culture period. Overall, the higher productivityin the control culture could be explained by a higher cell specificproductivity. Metabolites and pH profiles were routinely monitored on adaily basis, but did not show any differences between the cultures.

Example 4

It was further found that the combination of the novel amino acid ratiosin both basal medium and feed medium showed the best performance.Optimizing the amino acid ratios had not only an effect in basal medium(in batch mode), but also in the feed medium (fed-batch mode). Cellswere incubated in basal medium with optimized AA ratios or RPMI AAratios and fed with either feed medium with optimized AA ratios or RPMIAA ratios.

Example 4A

The impact of basal medium and feed medium was analyzed by culturingCHO2 (CHO-DG44) Rituximab cells in media with optimized amino acidratios (optimized AA ratios, medium 6.2 and feed 6.2) or non-optimizedamino acid ratios (RPMI AA ratios, medium 6.3 and feed 6.3) in afed-batch mode in all four combinations at a standard feed rate of 30ml/L/d based on the culture starting volume for all cultures. Basal andfeed medium 6.2 containing optimized AA ratios are further optimized forserum-free recombinant protein production and are chemically defined andare superior to the modified RPMI media used. In another experiment (in2-L bioreactor system, Example 4C), the final glucose concentration inthe feed solution (feed medium 6.2.1 and feed medium 6.3.1) wasincreased to minimize the number of glucose additions and operator workby adding stock solutions (Table 3).

Materials and Methods:

Basal medium 6.2 and medium 6.3 were identically designed comprisingabout 44 mM total amino acids, but different amino acid ratios (Tables 2and 2a). Likewise feed medium 6.2 and feed medium 6.3 were identicallydesigned comprising about 508-511 mM total amino acids, but differentamino acid ratios (Tables 3 and 6). In order to avoid an increasedosmotic pressure, the glucose concentration in the feed medium 6.2 and6.3 was reduced to a final concentration of 42 g/l. Glucose was furtheradded on demand to maintain glucose >1 g/L during the experimentalcourse.

The experiment was performed in a 48-miniaturized bioreactor system witha starting volume of 14 ml. In all cultures CHO2 (CHO-DG44) Rituximabcells were seeded at 0.3×10⁶ cells/ml in test medium or in controlmedium as follows: basal medium 6.2 and feed medium 6.2 (optimized AAratios, AA added as premixed powder), basal medium 6.3 and feed medium6.3 (RPMI AA ratios, AA added as premixed powder). The bioreactors wereincubated at 36.5° C. for the entire cultivation period and dissolvedCO2 was controlled between 2-15% to prevent toxic concentrations basedon the pH set-point of (7.20-6.80)+/−0.2.

TABLE 3 Composition of Feed Media 6.2 and 6.2.1 (with optimized AA) andMedia 6.3 and 6.3.1 (without optimized AA) Feed Feed Component (totalAA) 6.2/6.2.1* 6.3/6.3.1 Unit Total AA conc. 508 511 mM WFI 0.7 0.7 l/lNaHCO3 1.5 1.5 g/l AA premixed powder (optimized 71.38 g/l AA ratios) AApremixed powder (RPMI AA ratios) 77.63 g/l Feed medium 6 powder withoutAA** 12.57 12.57 g/l Insulin (5 g/L stock sol.) 10 10 ml/l (pharmaBiocon)*** Iron choline citrate (ICC; MW = 991.5 0.56 0.56 g/l g/mol)Dr. Paul Lohmann GmbH KG L-Ornithine × HCL 7.65 7.65 mg/l Putrescine ×2HCl [mg/l] 185.022 185.022 mg/l Glucose 35.4/58.4 35.4/58.4 g/lL-Glutamine 0 0 g/l Succinic acid 5.26 5.26 g/l 40% NaOH on on ml/ldemand demand WFI add 1.0 add 1.0 l/l Total glucose 42/65 42/65 g/l*Difference between feed medium 6.2 and 6.2.1 and feed medium 6.3 and6.3.1 is the total glucose content. **Feed medium 6 powder without AAcontains 6.6 g glucose. ***lnsulin may be substituted with IGF at afinal concentration of 250 μg/l

Results:

The effect of optimized amino acid ratios in basal medium and feedmedium for IgG1 antibody (Rituximab) production in a controlledmini-bioreactor system in fed-batch (n=2) are shown in FIG. 4C. Themaximal product concentration of 2786 mg/L on day 10 (2677 mg/L on day12) was achieved with optimized amino acid ratios in both, basal andfeed medium. Using a basal medium with RPMI AA ratios, but a feed mediumwith optimized AA ratios led to a considerably lower maximal productconcentration of 2126 mg/L on day 12. Productivity was even furtherdecreased in cultures using a basal medium with optimized AA ratios anda feed medium with RPMI AA ratios, resulting in a final titer of 1662mg/L on day 12. Lowest product concentration was achieved withnon-optimized amino acid ratios in both, basal and feed medium with aproduct concentration of 1577 mg/L on day 12.

Use of basal medium with non-optimized AA ratios followed by feed mediumwith optimized AA ratios slightly delayed viable cell concentrations,but reached almost comparable maximum viable cell concentrations.Compared to the respective cell culture using basal medium withoptimized AA ratios the cell specific productivity was also slightlyreduced (132.91 mg/10⁶ cells vs. 161.3 mg/10⁶ cells). Likewise theproductivity was slightly delayed, particularly in earlier days (days4-8) and remained lower over time. This shows that optimized AA ratiosare beneficial for cell productivity in both, the basal medium and thefeed medium.

This is accompanied with an increase in viable cell concentrations andviability for cultures in medium using optimized AA ratios, preferablyin both the basal and the feed medium. Maximal viable cellconcentrations for cell cultured in medium having optimized amino acidratios in basal medium and in feed medium were found to be 16.6×10⁶cells/ml on day 8. Culturing cells in a basal medium with RPMI AA ratiosand a feed medium with optimized AA ratios resulted in almost the samemaximal viable cell concentration of 16.0×10⁶ cells/ml (day 10),however, about two days later. Thus, non-optimized AA ratios in thebasal medium seem to delay viable cell proliferation. Culturing cells ina feed medium with RPMI AA ratios severely reduced the maximum viablecell concentration to 13.3×10⁶ cells/ml (basal medium with optimized AAratios) or 11.5×10⁶ cells/ml (basal medium with RPMI AA ratios) on day8. Thus, optimized AA ratios in the feed medium seem to support higherviable cell concentrations.

A similar trend was also observed for viability (FIG. 4B), with anearlier and more severe decrease in viability in cultures withoutoptimized AA ratios in the feed medium. No significant impact wasobserved for any of the other measured parameters.

In summary, the effect of a basal medium without optimized AA ratiosseems to result in a reduced cell specific productivity, which cannot betotally compensated by using an optimized feed medium. Use of feedmedium without optimized AA ratios on the other hand resulted in areduced number of viable cells (FIG. 4A) and viability (FIG. 4B). Thus,feed medium with optimized AA ratios improved viability and viable cellconcentration and thereby increased productivity, but also showed aneffect on cell specific productivity (161.2 mg/10⁶ cells vs. 124.9mg/10⁶ cells). In contrast to that, results for growth, viability andfinal titer also revealed that the maximal growth and maximal productconcentration were clearly impacted by optimized AA ratios in basalmedium and feed medium.

Example 4B

The impact of basal medium and feed medium was also analyzed usingoptimized amino acid ratios (optimized AA ratios, basal medium 6.2 andfeed 6.2) or non-optimized amino acid ratios (RPMI AA ratios, medium 6.3and feed 6.3) in a fed-batch mode in all four combinations at reducedfeed rates in an uncontrolled shake flask system (pH and dissolvedoxygen not controlled). The standard fed-batch feeding rate was adjustedfrom 30 ml/L/d (control) to 20 ml/L/d and 8 ml/L/d to avoid overfeedingand hence masking an effect.

Materials and Methods:

CHO2 (CHO-DG44) Rituximab cells were seeded at 0.3×10⁶ cells/ml in basaland feed medium 6.2 (optimized AA ratios) or in basal and feed medium6.3 (RPMI AA ratios). Feed medium 6.2 and feed medium 6.3 contained ametabolically adjusted glucose concentration of 42 g/l to ensurecomparable metabolic profiles (e.g. glucose) of shake flask experimentsand 2 L bioreactors. Viable cells, viability, product concentration,glucose concentration, lactic acid concentration, ammonium concentrationand osmolarity were measured as described above according to the sampleintervals. Experiments and controls were performed in duplicates (N=2).

In this experiment 500 ml shake flasks with a starting volume of 60 mlwere used to culture cells in basal and feed medium with or withoutoptimized AA ratios. The shake flasks cultures were incubated at 36.5°C. in an incubator (8% CO2 from day 0 to 2 and 5% CO2 from day 2, and 3%CO2 from day 3 until the end of the cultivation). Feed rate was set to20 ml/L/d for days 1 to 5 and 8 ml/L/d for days 5 to 11. Feed solutionwas added every 2 days to the culture with the aim to prevent glucoseoverfeeding and minimize osmotic pressure caused by an increased glucoselevel. The feed rate was calculated as follows e.g. 30 ml/L/d *0.06L=1.8 ml feed/day=3.6 ml feed/2 days, metabolically adjusted feed rate20 ml/L/d=1.2 ml/d=2.4 ml feed/2 days. Glutamine was maintained >0.1 g/Lover the cultivation, mainly replenished from an increased L-glutamineconcentration in the basal medium at start, but not from feed medium.

Results:

Effect of optimized amino acid ratios in medium and feed for IgG1antibody (Rituximab) production in uncontrolled shake flask system infed-batch mode at reduced feed rate (N=2).

The effect of basal medium can be seen if one compares the maximalproduct concentration of 897 mg/L (filled diamond) in cultures withnon-optimized amino acid ratios in basal medium 6.3 and optimized aminoacid ratios in feed medium 6.2 with 1049 mg/L (filled square) incultures with optimized amino acid ratios in basal medium 6.2 andoptimized amino acid ratios in feed medium 6.2, both at reduced feedrates (FIG. 4F). Having non-optimized amino acid ratios in the basalmedium therefore resulted in delayed and reduced product formation. Themaximal product concentration of 641 mg/L (filled circle) in cultureswith optimized amino acid ratios in basal medium 6.2 and withnon-optimized amino acid ratios in feed medium 6.3 was higher than themaximal product concentration of 468 mg/L (filled triangle) in cultureswith non-optimized amino acid ratios in basal medium and in feed medium(FIG. 4F). This result clearly demonstrates the positive impact ofoptimized amino acids in basal medium on maximal product titer.

Furthermore, the maximal product concentration of 1049 mg/L was achievedin cultures with optimized amino acid ratios in basal medium and feedmedium, which was reduced to 641 mg/L when using a feed medium withnon-optimized AA ratios at reduced feed rates (FIG. 4F). A similartendency for product production was found for cells cultured in basalmedium without optimized AA ratios and a feed medium with optimized AAratios (897 mg/L) or a feed medium without optimized AA ratios (468mg/L). This means that there is a strong positive impact of optimizedamino acids in feed medium on maximal product performance, however, bestresults were achieved when using optimized AA ratios in both the basaland the feed medium.

The viability profile followed a similar trend with a sharp decreasefrom 96% on day 5 for all cultures (FIG. 4E). The maximal viable cellconcentration ranged from 3.8-9.6 10⁶ cells/ml on day 5-6 (FIG. 4D). Ingeneral, a feed medium with optimized AA ratios increased viable cellconcentration (FIG. 4D). This result was in line with the improvedviability (FIG. 4E).

Furthermore, optimized amino acid ratios in the basal medium had apositive effect on cell proliferation. This may be taken from acomparison of the viable cell concentration for cells cultured in basalmedium with (7.27×10⁶ cells/ml, filled circle) or without (3.8×10⁶cells/ml, filled triangle) optimized amino acid ratios and feed mediumwithout optimized amino acid ratios on day 5. This result illustratesthe positive effect of an optimized basal medium on maximal growthperformance.

When feed medium with optimal AA ratios was used, the maximal viablecell concentration was comparable for a basal medium without optimizedAA ratios (9.6×10⁶ cells/m, filled diamond) and with optimized AA ratios(7.9×10⁶ cells/ml, filled square). The feed effect can be described ifone compares the maximal growth of cells cultured with optimized aminoacid ratios in basal medium and with optimized amino acid ratios in feedmedium (7.9×10⁶ cells/ml, filled square) or without optimized AA ratiosin the feed medium (7.2×10⁶ cells/ml, filled circle). Likewise, whenusing a basal medium without optimized AA ratios, the maximal viablecell concentration was 9.6×10⁶ cells/ml for cells cultured in feedmedium with optimized AA ratios and this was reduced to 3.9×10⁶ cells/mlfor cells cultured in feed medium without optimized AA ratios (FIG. 4D).

Generally, there are two major aspects that are in good agreement to theviable cell growth. First, the highest remaining viability of 37-40% wasachieved on day 9 for cultures with optimized amino acid ratios in feed(with or without optimized amino acid ratios in basal medium). Secondly,the viability drop down from day 5 onwards was shifted by approximatelyone day for the cultures with optimized amino acids in the feed medium.These results clearly show that a higher viability and a prolongation ofthe viability profile can be obtained with optimized basal medium andfeed medium.

Example 4C

The impact of basal medium and feed medium with and without optimizedamino acid ratios was further tested in a standard fed-batch format inan up-scaled fully controlled 2-L bioreactor system.

The 2 L bioreactor system is a representative model for large scalebioreactors for commercial manufacturing (up to 12,000 L scale andbeyond). The standard fed-batch feeding rate of 30 ml/L/d was appliedand feed solution was fed in a continuous mode starting from day 2 today 14. Other process parameters were set to our platform conditions forsuccessful scale-up based on our experience, i.e. oxygen transfer, shearforce, CO2 removal, pH range, agitation and power input per volume. Themedium combinations were tested in duplicates (N=2).

Materials and Methods:

The experiment was performed in a fully controlled 2 L bioreactor systemwith a starting volume of 1.8 L. CHO2 (CHO-DG44) Rituximab cells wereseeded at 0.3×10⁶ cells/ml in all cultures using basal medium 6.2(optimized AA ratios) or basal medium 6.3 (RPMI AA ratios) and feedmedium 6.2.1 (optimized AA ratios and adapted glucose concentration of65 g/l) or feed medium 6.3.1 (RPMI AA ratios and adapted glucoseconcentration of 65 g/l). The bioreactors were incubated at 36.5° C. forthe entire cultivation and dissolved CO2 was controlled between 2-15% toprevent toxic concentrations based on the pH set-point of (6.95 on days0-3 and 6.80 on days 3-day 14)+/−0.20.

Glucose concentration in the feed solution was optimized to a finalconcentration of 65 g/l in order to minimize osmotic pressure caused byglucose over feeding, but also to reduce the number of glucose additionsfrom stock solutions if necessary. The design of feed medium 6.2 andfeed medium 6.2.1 was identical except for the final glucoseconcentration. Likewise feed medium 6.3 and feed medium 6.3.1 wereidentical except for the final glucose concentration.

Viable cells, viability, product concentration, glucose concentration,lactic acid concentration, ammonium concentration and osmolarity weremeasured as described above according to the sample intervals. The feedmedia contained glucose, but no L-glutamine, thus glutamine was addedfrom a stock solution on demand to keep the glutamine concentration inthe range of 0.1-0.4 g/l. Glucose level was to be maintained at >2 g/Lfor the entire cultivation. Experiments were performed in duplicates(N=2).

Results:

In general, results of the 2 L system were in good agreement to thefindings from the previous shake flasks experiments with respect tomaximal titer and viable growth. For example, the maximal productconcentration of 2213 mg/L (filled squares) was achieved with optimizedamino acid ratios in basal medium and feed medium (FIG. 4I). Culturingcells without optimized amino acid ratios in the feed medium reduced themaximal product concentration to 1654 mg/L (filled circles) as shown inFIG. 4I. Culturing cells in basal medium without optimized amino acidratios strongly delayed product formation. A similar maximal productconcentration of 2213 mg/L (optimized amino acid ratios in basal andfeed medium) vs. 2144 mg/L (optimized amino acid ratios only in the feedmedium) was obtained due to the positive effect of optimized feedmedium. However, the product formation kinetics were clearly differentdue to the impact of non-optimized basal medium. Thus, for optimalproduct concentrations optimized amino acid ratios are required to bepresent in both, the basal and the feed medium.

These observations were in good agreement with the viable cellconcentrations. The maximal viable cell peak of 12.7×10⁶ cells/ml wasachieved with optimized amino acid ratios in basal medium and feedmedium compared to the maximal cell peak of 8.7×10⁶ cells/ml with eithernon-optimized amino acid ratios in basal medium or feed medium (FIG.4G). The maximal viable growth peak of 12.7×10⁶ cells/ml was due to acombined effect on viable cell concentration of optimized amino acidratios in basal medium and feed medium for fed-batch cultures. Further,the effect of basal medium and the effect of feed can be seen if onecompares the exponential growth phase from day 4-9 for the cultureswithout optimized AA in basal medium and with optimized AA in feedmedium vs. a culture with optimized AA in basal medium and withoutoptimized AA in feed medium. FIG. 4G shows that the growth kinetic foroptimized basal medium was steeper compared to non-optimized basalmedium although both cultures achieved a similar maximal cell peak ofapproximately 8.6×10⁶ cells/ml. In contrast, the slower growth kineticwith non-optimized basal medium, but optimized feed medium led to aprolongation of viable cells from day 10 to 14.

The viability profile followed a similar trend as discussed above. Amaximal viability over a prolonged run time could be attributed to thefeed effect (compare the viability of 79% vs. 49-54% on day 14) (FIG.4H). Viability profiles for cells with optimized AA ratios in basalmedium and in feed medium or with optimized AA ratios in basal mediumand without optimized AA ratios in feed medium followed a similar trend.Other measured parameters such as metabolites and pH did not show anysignificant differences.

Example 4D

The impact of optimized AA ratios in basal medium and feed medium wasfurther investigated (optimized AA ratios in RPMI basal medium 3.9 andRPMI feed medium 3 or non-optimized amino acid ratios RPMI AA ratios inRPMI basal medium 3.1 and RPMI feed medium 2) in a RPMI environment infed-batch mode for all four combinations using CHO2 (CHO-DG44) Rituximabcells. RPMI is a commercial medium with a known composition.

The total amino acid concentration in RPMI basal cell culture medium andRPMI feed medium increased with adjusting the amino acid ratio to theoptimized amino acid ratio of the invention. To rule out that theobserved effects were simply due to an increased overall amino acidconcentration, in a separate experiment RPMI basal cell culture mediumand RPMI feed medium was adjusted with different amino acid ratios(spent media optimized amino acid ratio).

Material and Methods:

The RPMI basal medium used in this experiment is based on thecommercially available RPMI medium R8755 (Mediatech catalog no. 90022PBor Sigma Aldrich catalog no. R8755) that was originally developed atRoswell Park Memorial Institute in 1966 by Moore and his co-workers(SAFC, Biosciences product information). For serum-free use it has beensupplemented as shown in table 4.

This experiment was performed in 250 ml shake flasks with a startingvolume of 100 ml. All cultures were seeded in shake flasks at 0.3×10⁶cells/ml in the specific media compositions: control RPMI medium 3.1(without optimized AA ratios, total AA 10.0 mM), RPMI medium 3.9 (withoptimized amino acid ratios, total AA−15.2 mM), RPMI feed medium-2(without optimized amino acid ratios, total AA 124 mM), RPMI feedmedium-3 (with optimized amino acid ratios, total AA 548 mM), RPMImedium 3.5 (RPMI medium 3.1+AA, spent media optimization, total AA 12mM), RPMI feed medium 3.5 (RPMI feed-2+AA, spent media optimization,total AA 140 mM). The 7 amino acids added for spent media optimizationof the basal medium were L-methionine, L-phenylalanine, L-proline,L-threonine, L-tryptophan, and L-tyrosine×2Na×2H₂O and L-valine eachadded at 30 mg/l and the amino acids added for spent media optimizationof the feed medium were L-cysteine, L-methionine, L-proline,L-threonine, L-tyrosine×2Na×2H₂O and L-valine each added at 360 mg/l.Basal medium 3.1, 3.5 and 3.9 was fortified with plant hydrolysate tosupport initial growth at the beginning of the cultivation. For thisreason the hydrolysate was not provided in the feeding solutions. As maybe taken from the experiments above (see medium 4 and medium 5) basalRPMI based medium may also be used without Hypep.

Shake flasks were incubated at 36.5° C. in an incubator (5% CO2atmosphere was provided from day 0 to 3 followed by 3% CO2 until the endof the cultivation). Feeding solution was added every second day at afeed rate of 30 ml/L/d from day 2-4 and at a reduced feed rate of 3ml/L/d from day 5-8. Glucose was fed on demand to maintain the actualglucose concentration between 2-4 g/l over the cultivation period. Totalcells, viable cells, viability, product concentration, glucoseconcentration, lactic acid concentration, ammonium concentration andosmolarity were measured every second day until the end of cultivationto monitor and control the experimental progress. Experiments wereperformed in duplicates (N=2).

TABLE 4 Composition of RPMI Basal Medium 3.1 (non-optimized AA), 3.9(optimized AA), 3.5 (spent media analysis) Components Medium 3.1 Medium3.9 Medium 3.5 Unit WFI 0.800 0.800 0.800 l/l RPMI 1640 (Product No.Sigma-Aldrich 10.40 10.40 10.40 g/l R8755) AA supplementation for spendmedia — 0.21 g/l analysis (met, phe, pro, thr, trp, tyr, val) AAsupplementation - optimized ratios 0.8883 NaHCO₃ 3.0 3.0 3.0 g/lMonoethanolamine (12.216 g/l stock 800 800 800 μl/l sol.) Sigma-AldrichChemie Iron choline citrate (ICC; 991.5 g/mol) 0.2 0.2 0.2 g/l Dr. PaulLohmann GmbH KG Fe-Citrate (10 g/l stock sol) 0.0 0.0 0.0 ml/l Selenicacid (25.79 mg/l stock sol.) 100.0 100.0 100.0 μl/l Putrescine × 2HCl4.8 4.8 4.8 mg/l Insulin (5 g/l stock sol.) 2 2 2 ml/l chem. definedLipids (Gibco Life 5.0 5.0 5.0 ml/l Technol. 92_0239DK) Hepes 3.57 3.573.57 g/l Glucose 1.50 2.17 1.50 g/l L-Glutamine total 0.85 0.70 0.85 g/lPluronic 1.00 1.00 1.00 g/l HyPep 1510 (Kerry Sheffield) 4.0 4.0 4.0 g/l40% NaOH as needed as needed as needed ml/l (adjust to pH = (adjust topH = (adjust to pH = 7.1 +/− 0.1) 7.1 +/− 0.1) 7.1 +/− 0.1) WFI add 1.0L add 1.0 L add 1.0 L l/l

TABLE 4a Amino Acid Ratios of Basal Medium RPMI 1640 (original), BasalMedium 3.1, 3.9, Medium 3.5 (spent media analysis), and Medium 6.2Medium Medium Medium Medium Amino Acid RPM11640 3.1 3.9 3.5 6.2 Total AAconc. 6.3 10.0 15.2 12 44 mM mM mM mM mM L-Alanine — — — — — L-Arginine2.5 2.5 2.5 2.5 2.1 L-Asparagine 1.0 1.0 1.8 1.0 1.8 L-Aspartic acid 0.40.4 1.3 0.4 1.3 L-Cysteine 1.0 1.0 2.2 1.0 1.6 L-Glutamic acid 0.4 0.40.9 0.4 0.9 L-Glutamine 5.4 15.3 12.6 15.3 46.4 L-Glycine 0.4 0.4 0.40.4 24.7 L-Histidine 0.2 0.2 0.9 0.2 0.9 L-Isoleucine 1.0 1.0 1.0 1.01.0 L-Leucine 1.0 1.0 1.7 1.0 1.7 L-Lysine 0.6 0.6 2.3 0.6 2.2L-Methionine 0.3 0.3 0.5 0.8 0.5 L-Phenylalanine 0.2 0.2 0.7 0.7 0.7L-Proline 0.5 0.5 2.3 1.1 2.3 L-Serine 0.8 0.8 2.1 0.8 2.1 L-Threonine0.4 0.4 1.5 1.1 1.5 L-Tryptophan 0.1 0.1 0.4 0.5 0.4 L-Tyrosine 0.3 0.32.1 0.6 2.1 L-Valine 0.5 0.5 1.5 1.1 1.5

TABLE 5 Composition of RPMI Feed Medium-2 (non-optimized AA), -3(optimized AA), -3.5 (spent media analysis) Component Feed-2 Feed-3Feed-3.5 Unit WFI 0.700 0.700 0.700 l/l RPMI Feed premix 83480CP 41.5841.58 41.58 g/l (w/o L-GIn, L-Cys and cystine, with RPMI AAs) 12xL-Cysteine × HCl × H₂O 3.12 2.60 3.12 g/l Cystine × 2 HCl 390.90 390.90750.90 mg/l AA supplement for feed — 61.75 — g/l medium-3 (optimized AA)AA supplement for spend media — — 1.80 g/l analysis (met, pro, trp, tyr,val) Insulin (5 g/l stock sol.) 10 10 10 ml/l Fe-citrate (10 g/l stocksol) 25.00 25.00 25.00 ml/l Selenic acid (25.79 mg/l stock sol.) 100.00100.00 100.00 μl/l L-Glucose 26.00 26.00 26.00 g/l L-Glutamine 8.00 8.008.00 g/l 40% NaOH on on on ml/l demand demand demand WFI add 1.0 add 1.0add 1.0 l/l Total Glucose 50.00 50.00 50.00 g/l Total Glutamine 8.008.00 8.00 g/l

TABLE 5a RPMI Feed premix (1x) 83480CP, without bulk salts*: COMPONENT[mg/L] COMPONENT [mg/L] L-Arginine 200 L-Lysine × HCl 40 L-Asparagine ×H2O 56.8 L-Methionine 15 L-Aspartic Acid 20 Niacinamide 1 D-Biotin 0.2L-Phenylalanine 15 D-calcium 0.25 L-Proline 20 pantothenate CholineChloride 3 PABA (Para- 1 aminobenzoic acid) Cyanocobalamin 0.005Pyridoxine × HCl 1 D-Glucose (dextrose 2000 Riboflavin 0.2 anhyd.)**Folic acid 1 L-Serine 30 L-Glutamic acid 20 Sodium phosphate 800(dibasic) L-Glutathione, reduced 1 Thiamine × HCl 1 L-Glycine 10L-Threonine 20 L-Histidine 15 L-Tryptophan 5 Hydroxy L-proline 20L-Tyrosine 2Na × 2H₂0** 14.4 myo-inositol 35 L-Valine 20 L-Isoleucine 50L-Leucine 50 Sum mg/L 3464.4 *Omitted bulk salts: Calcium dinitrate ×4H₂O, magnesium sulfate, potassium chloride, sodium chloride and sodiumhydrogen carbonate **Added separately

TABLE 6 Amino Acid Ratios of RPMI Feed-2 (non-optimized) and RPMI Feed-3(optimized AA), RPMI Feed Medium 3.5 (spent media analysis), Feed Medium6.2 and 6.2.1 (optimized AA) and Feed Medium 6.3 and 6.3.1(non-optimized AA) Feed Feed Feed Feed Feed Amino Acid 2 3 3.5 6.2/6.2.16.3/6.3.1 Total AA conc. 124 548 140 508 511 mM mM mM mM mM L-Alanine —— — — — L-Arginine 3.01 0.97 3.01 0.97 2.49 L-Asparagine 0.99 3.22 0.993.22 0.87 L-Aspartic Acid 0.39 0.23 0.39 0.23 0.39 L-Cysteine 4.44 0.804.94 0.68 0.34 L-Glutamic Acid 0.36 0.26 0.36 0.26 0.36 L-Glutamine11.97 2.55 11.97 — — L-Glycine 0.35 0.29 0.35 1.12 0.76 L-Histidine 0.251.73 0.25 0.57 0.25 L-Isoleucine 1.00 1.00 1.00 1.00 1.00 L-Leucine 1.003.22 1.00 3.22 1.00 L-Lysine 0.57 1.70 0.57 1.60 0.57 L-Methionine 0.260.58 0.79 0.58 0.26 L-Phenylalanine 0.24 0.86 0.24 0.86 0.24 L-Proline0.46 1.35 1.14 1.35 0.46 L-Serine 0.75 3.23 0.75 3.23 0.75 L-Threonine0.44 1.84 1.10 1.84 0.44 L-Tryptophan 0.06 0.45 0.06 0.45 0.06L-Tyrosine 0.14 0.03 0.45 0.83 0.41 L-Valine 0.45 1.57 1.12 1.57 0.45

In the spent medium analysis commercial available RPMI medium wasmodified and fortified with various nutrient supplementations to avoidnutrient limitations and to ensure improved growth and product formationin the fed-batch experiment. For this purpose AA supplements were addedto the medium according to the medium recipe for medium 3.1. An aminoacid analysis was performed for samples taken from the cell culturesupernatant on days 4 and 7, except for L-arginine. As a result it wasfound that the concentration of the following seven amino acids wasbelow 15 mg/L: L-valine, L-threonine, L-proline, L-methionine,L-phenylalanine, L-tyrosine and L-tryptophan. Based on this prior artspent media analysis, these amino acids were additionally supplementedin a basal medium (RPMI medium 3.5). Specifically, the amino acidsL-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan,L-tyrosine, and L-valine were additionally provided each at 30 mg/L inthe basal medium (Tables 4 and 4a). In the feed medium, the amino acidsL-methionine, L-threonine, L-proline, L-cystine, L-tyrosine and L-valinewere additionally provided each at 360 mg/L (Tables 5 and 6). Theexperiment was basically performed as described above using RPMI basalcell culture medium with or without optimized amino acid ratios and aRPMI feed medium with and without optimized amino acid ratios.Additionally cells were incubated with RPMI basal medium and feed mediumcomprising spent media amino acid ratios in either the RPMI basal cellculture medium or in the RPMI feed medium or in both.

Results:

In summary, the effect of the optimized AA ratios in RPMI basal cellculture medium and/or in RPMI feed medium on cell culture performance infed-batch mode was investigated using CHO2 (CHO-DG44) cells producingthe antibody Rituximab. Specifically, cell viability (FIG. 4L, O),viable cells (FIG. 4K, N), product titer (FIG. 4M, P) and lactateconcentration (data not shown) were monitored.

The major effect of optimized feed medium or basal medium could be seenin the final product concentration and also in the product kinetics(slope of curve) as indicated in FIG. 4M.

For example, the product titer of cells cultured in RPMI basal cellculture medium and RPMI feed medium both comprising the optimized aminoacid ratios were higher (259 mg/L) compared to control cells cultured inRPMI basal cell culture medium and RPMI feed medium without optimizedamino acid ratios (162 mg/L), as shown in FIG. 4M. In a culture withoptimized AA ratios in the basal medium, but non-optimized AA ratios inthe feed medium, the maximal product titer was reduced to 171 mg/ml.Interestingly, the effect of optimized amino acid ratios in the feedmedium was similar up to day 4 (167 mg/L vs. 161 mg/L) and only differedat later culture days. Furthermore, in the case of non-optimized basalmedium, the product formation and curve kinetic (slope of the curve) wasdelayed and resulted in a maximal product concentration of approximately152-162 mg/L on day 8 (without optimized AA ratios in basal medium andwith or without optimized AA in feed medium).

A similarly positive effect could be observed for the viable cellconcentration and viability using the RPMI media system (RPMI basal cellculture medium and RPMI feed medium). Highest viable cell concentrationswith a maximal viable cell concentration of approximately 3.5×10⁶cells/ml (FIG. 4K) and highest cell viabilities (FIG. 4L) were achievedwhen the optimized amino acid ratios were used in both, RPMI basal cellculture medium and RPMI feed medium. If the optimized amino acid ratioswere only applied in the basal cell culture medium a sharp decrease inviable cell concentration (FIG. 4K) and viability (FIG. 4L) was observedfrom day 6 onwards. The maximal viable cell concentration was lower(FIGS. 4K and N) for cells cultured in basal cell culture medium withoutoptimized AA ratios and feed medium with optimized AA ratios (2.2×10⁶cells/ml) and even lower for cells cultured in basal cell culture mediumand feed medium, both without the optimized amino acid ratios (maximalviable growth up to 1.70 10⁶ cells/ml, day 4). In summary, this exampledemonstrates the superiority of the optimized amino acid ratio on cellculture performance in a RPMI based medium, namely product titer, viablecell concentrations and cell viability.

Similar results were obtained in the other experiment including the samemedia and a medium supplemented with AAs according to the spent mediaanalysis.

As mentioned above, adjusting the optimized amino acid ratios in RPMIbasal cell culture medium significantly improved product titers comparedto the unmodified RPMI media system. Product titer in cell culturecomprising the optimized amino acid ratios in both, basal and feedmedium was higher compared to control without any implementation ofoptimized amino acid ratios in RPMI (titer of 0.406 g/L vs. 0.173 g/L).Hence by adjusting amino acid ratios according to the optimized aminoacid ratios in both basal medium and feed medium, the product titer wasincreased by a factor of about 2.3 in a commercial media system such asRPMI. Product titer in RPMI basal cell culture medium comprisingoptimized amino acid ratios and RPMI feed medium without amino acidadjustment was higher compared to the controls without implementation ofoptimized amino acid ratios in basal and feed medium (titer of 0.267 g/Lvs. 0.173 g/L). Product titer in a culture with optimized amino acidratios only in the RPMI feed medium was almost comparable to the controlwithout any novel amino acid ratios implemented in either basal mediumor feed medium (titer of 0.159 g/L vs. 0.173 g/L). Again, this resultdemonstrates that the optimized amino acid ratios should be applied fromthe beginning of a cultivation experiment, i.e. in both basal and feedmedium. In this setting, application of optimized amino acid ratios onlyin feed medium was not sufficient to achieve maximal product titers.

The product titer in RPMI with spent media amino acid ratios in bothbasal medium and feed medium was higher (0.302 g/L, open square)compared to the control without any amino acid ratio adjustment (0.173g/L, filled diamond), but lower compared to the optimized amino acidratios in RPMI medium and in RPMI feed medium (0.406 g/L, filledsquare). Furthermore, product titer with spent media amino acidadjustment in RPMI basal medium, but not in RPMI feed medium (0.193 g/L,open circle) was higher compared to the control without optimized aminoacid ratios in basal or feed medium (0.173 g/L, filled diamond), butclearly lower compared to the optimized amino acid ratios in RPMI basalculture medium only (titer 0.267 g/L, filled circle) (FIG. 4 P).

Thus the effect of spent media amino acid ratio adjustment in basal cellculture medium and in feed medium (maximal titer 302 mg/L) was reducedcompared to the impact on overall cell culture performance of theoptimized amino acid ratios in basal cell culture medium and in feedmedium (maximal titer of 406 mg/L). For spent media amino acid ratioadjustment only in the basal medium, but not in the feed medium amaximal titer of only 193 mg/L was obtained.

According to the titer, the viable cell concentration that is achievedfor all cultures follows a similar trend with a maximal cell peak on day4. Most cultures have a maximal viable cell concentration ofapproximately 3.5×10⁶ cells/ml, except for cultures without anysupplementation in the basal medium (1.7-2×10⁶ cells/ml). The viablegrowth with highest viable cell numbers over time was achieved withcells in optimized amino acid ratios in both basal medium and feedmedium. This result demonstrates a combined effect of optimized basalmedium and optimized feed as shown in FIGS. 4N and P.

The viability profile (FIG. 4O) follows a similar trend with a breakdownon day 4 for most of the cultures except for the culture with optimizedAA ratios in basal medium and feed medium. This finding is in goodagreement to the viable growth pattern as shown in FIG. 4N. Nosignificant impact of the media on other parameters such as metabolitesand pH was observed.

Example 5

It was found that certain amino acids have an impact on cell metabolismwith respect to the maximal product concentration, viable cellconcentration and viability. The impact of varying these amino acids wasfurther analyzed in combination in fed-batch mode with a serum-free,chemically defined medium. The variation of the amino acids wasinvestigated within two AA groups that are a) L-phenylalanine, L-valine,L-leucine, L-threonine, L-isoleucine (5 AAs) and b) L-phenylalanine,L-valine, L-leucine, L-threonine, L-isoleucine, L-tyrosine, L-lysine (7AAs). The amino acids were then varied in basal medium and feed mediumby +/−20% and +/−40% in a positive or negative alternating mode based onthe optimized amino acid ratios from the control (basal medium 6.4.0.1and feed medium 6.4). Alternating mode means that the first AA isincreased, the second AA is decreased, the third AA is increased etc. inthe same direction in basal medium and in feed medium by 20% or 40%(calculated on a molar basis). The alternating mode is described by theusage of small letters (reduction by −20% or −40%, e.g. his, tyr) andcapital letters (increase by +20% or +40%, e.g. HIS, TYR). In order toprovoke a strong cellular response with respect to maximal growth andproduct formation, the nutrient feeding rates was reduced in someexperiments.

Materials and Methods:

CHO2 (CHO-DG44) Rituximab cells were cultured in fed-batch in medium6.4.0.1 and feed medium 6.4 (with optimized amino acid ratios) at astandard feed rate and a reduced feed rate. The experiment was separatedinto 3 approaches testing AA variations in combination that are a)variation of 5 AAs in basal medium and feed medium by +/−40%, b)variation of 7 AAs in basal medium and feed medium by +/−40%, and c)variation of 7 AAs in basal medium and feed medium by +/−20% or +/−40%at a reduced feed rate.

For the 5 AA set-up the amino acids L-phenylalanine, L-valine,L-leucine, L-threonine, L-isoleucine were varied by +/−40% in a positiveor negative alternating mode (capital or non-capital AA letters)compared to control with optimized amino acid ratios in both basal andfeed medium. Media used were: Basal medium 6.4.3 (5 amino acids PHE,val, LEU, thr, ILE varied by +/−40%, positive), basal medium 6.4.4 (5amino acids phe, VAL, leu, THR, ile varied by +/−40%, negative), feedmedium 6.4.3 (5 amino acids PHE, val, LEU, thr, ILE varied by +/−40%,positive), feed medium 6.4.4 (5 amino acids phe, VAL, leu, THR, ilevaried by +/−40%, negative).

For the 7 AAs set-up the following media were used: Basal medium 6.4.5(7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by +/−20%,positive), basal medium 6.4.7 (7 amino acids PHE, val, LEU, thr, ILE,tyr, LYS varied by +/−40%, positive), basal medium 6.4.8 (7 amino acidsphe, VAL, leu, THR, ile, TYR, lys varied by +/−40%, negative), feedmedium 6.4.5 (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS varied by+/−20%, positive), feed medium 6.4.7 (7 amino acids PHE, val, LEU, thr,ILE, tyr, LYS varied by +/−40%, positive), feed medium 6.4.8 (7 aminoacids phe, VAL, leu, THR, ile, tyr, lys varied by +/−40%, negative). Itshould be added that due to solubility reasons L-tyrosine was notincreased in the alternated mode of feed medium 6.4.8, which was onlyrelevant for a 7 AA variation by +40% not for a 20% AA variation. Exceptfor feed medium 6.4.8, lacking the increased tyr concentration, thevariations of the amino acids used in the feed medium were the same asin the basal medium in all cultures.

The experiment was performed in a miniaturized bioreactor system with astarting volume of 14 ml. In all cell cultures CHO2 (CHO-DG44) Rituximabcells were seeded at 0.3×10⁶ cells/ml in basal medium for fed-batchcultivation. The bioreactors were incubated at 36.5° C. for the entirecultivation and dissolved pCO2 was controlled between 2-15% to preventtoxic concentrations based on the pH set-point of (7.20-6.80)+/−0.2. Thestandard feed rate of 30 ml/L/d was applied to cultures with a 5 AAvariation by +/−40% (FIG. 5A-C) and 7 AA variation by +/−40% (FIG.5D-F). A reduced feed rate of 20 ml/L/d on days 1-5 and 8 ml/L/d on days6-11 was applied to cultures with 7 AA variation by +/−20% and +/−40%(Figure G-I). Feed solution was added continuously to the culture andattention was paid to prevent glucose overfeeding and minimize osmoticpressure caused by an increased glucose addition. For example, feedmedium 6.4.3, feed medium 6.4.4, feed medium 6.4.7 or feed medium 6.4.8contained a reduced glucose concentration of 42 g/l. All feed mediacontained glucose, but no L-glutamine, thus glutamine was added from astock solution on demand to keep the glutamine concentration in therange of 0.1-0.4 g/l. Glucose was also added on demand to keep theglucose level >2 g/L for the entire cultivation. Viable cells,viability, product concentration, glucose concentration, lactic acidconcentration, ammonium concentration and osmolarity were measured asdescribed above according to the sample intervals. Experiments andcontrols were performed in duplicates (N=2).

Results:

The combined variation of 5 AAs or 7 AAs (+/−40% at a standard feedrate) and especially the combined variation of 7 AAs at a reduced feedrate (7 AAs+/−20% and +/−40% at a reduced feed rate) in a medium withotherwise optimal AA ratios reduced the productivity and viable growthin cell cultures.

Variation of 5 AAs: The highest maximal product concentration of 1909mg/L was achieved on day 10 in control cultures (optimized AA ratios inbasal and feed medium) (FIG. 5C). For variations of 5 AAs, the maximalproduct concentration was reduced by alternately varying 5 AAs in bothdirections to 1523 mg/L (phe, VAL, leu, THR, ile+/−40%, alternatingmode) and 1799 mg/L (PHE, val, LEU, thr, ILE+/−40%, alternating mode),respectively. Interestingly, the product formation showed a differentkinetic (slope of curve), as may be seen from the product titer on day10 with 1909 mg/L (control) vs. 1523 mg/L vs. 1498 mg/L (PHE, val, LEU,thr, ILE and phe, VAL, leu, THR, ile, respectively, +/−40%, alternatingmode). A combined variation of 5 AAs by +/−20% showed a maximal productconcentration that was only slightly reduced to (phe, VAL, leu, THR, Ilevaried by +/−20%, 1806 mg/l on day 10) or comparable to (PHE, val, LEU,thr, ILE varied by +/−20%, 2058 mg/L on day 12) control cultures (datanot shown). The results indicate that the variation of five amino acidsin combination by only +/−20% had no significant impact on productformation or on the maximal product titer.

The viable cell concentration profile followed a similar trend for allcultures (control versus test cultured varied by +/−40%) with a maximalviable peak density in the range of 13.8-19.4×10⁶ cells/ml on day 8(FIG. 5A). A maximal viable cell density of 19.4×10⁶ cells/ml wasobserved in control cultures with optimized AA ratios in basal medium(basal medium 6.4.0.1) and feed medium (feed medium 6.4). The maximalviable cell concentration in cultures with varied amino acid ratios wereconsiderably lower (PHE, val, LEU, thr, ILE by +/−40%, viable cellconcentration of 13.8×10⁶ cells/ml; phe, VAL, leu, THR, ile+/−40%,viable cell concentration of 15.3×10⁶ cells/ml) (FIG. 5A). The viablegrowth of the test cultures with varied amino acids by +/−20% (5 AA) wascomparable to the control cultures with optimized amino acid ratios(data not shown).

Furthermore, the viability profile of all cultures was fairly comparableshowing a decline in viability for all cultures starting on day 8.Interestingly, in one of the cultures with the amino acid ratios of 5AAs varied by +/−40% (PHE, val, LEU, thr, ILE), the viability remainedrather high at 56% at the end of the cultivation period (days 11-14),compared to a viability of 13% for the control culture on day 14 (FIG.5B). For test cultures with the amino acid ratios of 5 AAs varied by+/−20% all curves were comparable to control (data not shown).

Variation of 7 AAs: Similar results were obtained with the variation of7 amino acids (L-phenylalanine, L-valine, L-leucine, L-threonine,L-isoleucine, L-tyrosine, L-lysine) by +/−40%. For example, the productconcentration of 1618 mg/L (phe, VAL, leu, THR, ile, tyr, lys by +/−40%,alternating mode) or 1456 mg/L (PHE, val, LEU, thr, ILE, tyr, LYS by+/−40%, alternating mode) on day 10 was reduced compared to the maximalproduct concentration of 1909 mg/L measured in control cultures on day10 (FIG. 5F). This result was in good agreement with the resultsobtained with the 5 AA variations. Altering 7 AAs by +/−20% resulted ina comparable maximal product concentration of 1861 mg/l (7 AA PHE, val,LEU, thr, ILE, tyr, LYS varied by +/−20%) or a slightly reduced maximalproduct concentration with 1622 mg/l (7 AA phe, VAL, leu, THR, ile, tyr,lys varied by +/−20%, negative alternating mode) compared to controlcultures with a maximal product concentration of 1909 mg/l on day 10(data not shown). This means that the variation of 7 amino acids by+/−20% only had a minor effect on product formation and maximal productconcentration in a cell culture.

The maximal viable cell concentration ranged from 14.4-19.4×10⁶ cells/mlon day 8 (FIG. 5D). The viable growth of the test cultures with 7 variedamino acids was comparable to the viable growth of the cultures withmedium comprising 5 varied amino acids varied by +/−40% (compare FIGS.5A and D). Likewise, the viability for 7 AA varied by +/−40% wascomparable to the cultures with 5 AA varied by 40% (compare FIGS. 5B and5E). Again one culture (7 amino acids PHE, val, LEU, thr, ILE, tyr, LYS,varied by +/−40%,) showed a higher viability of 45% compared to controlat the end of cultivation.

Altering 7 AAs by +/−20% resulted in a comparable growth profile for allcultures including control cultures with a similar maximal peak celldensity on day 8 of 18.9-20.1×10⁶ cells/ml (data not shown). Also theviability profile was comparable for all cultures that remained ratherhigh with 95% until culture day 8, but then dropped below 30% at the endof the culture period (data not shown).

Variation of 7 AA with reduced feed rate: To potentiate the effect cellswere additionally cultured in a fed-batch mode using a reduced feedrate. Compared were control cultures with a standard feed rate (medium6.4.0.1 and feed medium 6.4, standard feeding), control cultures withreduced feed rate (medium 6.4.0.1 and feed medium 6.4 with reduced feedrate) and test cultures with 7 amino acids varied by +/−20% or +/−40% atreduced feed rate. The maximal product concentration in control culturesat a standard feed rate was 1909 mg/l on day 10 (1782 mg/L day 12,filled square) and 1611 mg/l for the control culture with a reduced feedrate (day 12, filled circle) (FIG. 5I). Altering the concentration of 7amino acids by 20% (PHE, val, LEU, thr, ILE, tyr, LYS; reduced feedrate) resulted in a maximal product concentration of 1448 mg/l (day 12,filled cross). This titer was further reduced in cultures with 7 AAsvaried by +/−40% to a maximal product concentration of 1269 mg/L (phe,VAL, leu, THR, ile, Tyr, lys; reduced feed rate, filled triangle) or 999mg/L (PHE, val, LEU, thr, ILE, tyr, LYS; feed rate reduced, filled X) onday 12. Thus, the variation of the AA ratio of 7 key amino acids reducedthe productivity compared to control culture and this was morepronounced when the feed medium was added at a reduced feed rate.

The viable cell concentration showed a comparable trend irrespective ofthe feed rate (compare FIGS. 5D and 5G). The viable cell concentrationshowed a maximal cell peak between days 6 and 8 (FIG. 5G). For example,control cultures showed a maximal viable cell concentration of 19.4×10⁶cells/ml at standard feed rate and this was slightly reduced at areduced feed rate to 16.5×10⁶ cells/ml. The maximal cell concentrationfor cultures with 7 AAs varied by +/−20% (PHE, val, LEU, thr, ILE, tyr,LYS) was approximately 13.5×10⁶ cells/ml and even lower with about11×10⁶ cells/ml for cultures with 7 AAs varied by +/−40%.

The viability profile for all cultures followed a similar trend with aclear decrease starting between days 8 and 10 (FIG. 5H). Otherparameters such as glucose, osmolarity or pH progress did not show anysignificant differences compared to the control cultures.

Example 6

In this fed-batch experiment, the impact of an optimized medium and feedmedium on the cell culture performance was demonstrated for severalCHO-DG44 cell lines that produce different monoclonal antibodies or afusion protein as an example for pharmaceutically relevant proteins. Theintention is to demonstrate that the optimized cell culture medium (withoptimized amino acid ratios in basal medium and with optimized aminoacid ratios in feed medium) clearly contributes to an improvedproductivity for a multipurpose manufacturing site.

Materials and Methods:

This experiment was performed in a miniaturized bioreactor system with astarting volume of 15 ml. All CHO-DG44 cell lines expressing a differenttherapeutic molecule were seeded at 0.3×10⁶ cells/ml in basal medium 6.2and feed medium 6.2, both with optimized AA ratios. The therapeuticmolecules expressed in CHO-DG44 cells were Rituximab with a heavy chainhaving the amino acid sequence of SEQ ID NO: 1 and a light chain havingthe amino acid sequence of SEQ ID NO:2, mAb6 with a heavy chain havingthe amino acid sequence of SEQ ID NO: 3 and a light chain having theamino acid sequence of SEQ ID NO: 4, mAb5 and an Fc-fusion proteinhaving the amino acid sequence of SEQ ID NO: 5. The bioreactors wereincubated at 36.5° C. for the entire cultivation and dissolved CO2 wascontrolled between 2-15% to prevent toxic concentrations based on the pHset-point of 6.95 (+/−0.15) and 6.80+/−0.15 from day 3 onwards. For thisfed-batch application a platform process for successful scale-up wasapplied that included a standard feed rate of 30 ml/L/d. This means thatthe nutrient feed solution was added daily for the entire cultivationfrom day 1 until the end of cultivation. Glutamine was added from astock solution on demand to keep the glutamine concentration in therange of 0.1-0.4 g/l. Glucose was also added on demand to keep theglucose level >0.6 g/L for the entire cultivation. Viable cells,viability, product concentration, glucose concentration, lactic acidconcentration, ammonium concentration and osmolarity were measured asdescribed above. Experiments and controls were performed in duplicates(N=2).

Results:

The product concentration of several CHO-DG44 cell lines expressingdifferent therapeutic proteins was high for all proteins, but variedslightly. The maximal product concentration varied from 8213 mg/L on day11 for mAb6 producing cells (FIG. 6C), 4655 mg/L on day 11 for mAb5producing cells (FIG. 6C), 1778-2061 mg/L on day 11 for Fc-fusionprotein and Rituximab producing cells (FIG. 6D). This variation in titerranging from 1.7 g/l up to >8.2 g/l is accompanied by a variable viablecell concentration and viability. These results demonstrate thatdifferent CHO-DG44 cells expressing a variety of different recombinantproteins were able to grow and proliferate in the optimized culturemedium in fed-batch mode (FIG. 6A-D).

Example 7

The effect of different concentrations of iron choline citrate on cellculture performance, specifically cell growth and product formation, wasinvestigated in shake flask experiments using medium 6.2a. It was foundthat (i) iron choline citrate increased product titers and (ii) that thenovel compound iron choline citrate is superior compared to commonlyused iron carriers such as, for example, iron pyro phosphate, ironphosphate and iron citrate.

Materials and Methods:

The experiment was performed in 250 ml shake flasks with a startingvolume of 100 ml. All cultures were seeded at 0.3×10⁶ cells/ml (CHO2(CHO-DG44) cells producing Rituximab) in basal medium 6.2a containingiron choline citrate at three different concentrations (0.2 g/l, 1 g/Lor 2 g/l) or iron pyro phosphate (0.5 g/l, 0.8 g/l or 1.3 g/l), or ironphosphate (0.3 g/l, 0.5 g/l, 0.7 g/l) at about equimolar amounts andfeed medium 6.2a without iron choline citrate. The concentration rangesof iron pyro phosphate and iron phosphate were chosen to be in the samerange (on a molar basis) as iron choline citrate. For example, ironcholine citrate at 1.0 g/L (titer of 2.81 g/L) is about equimolar toiron phosphate at 0.3 g/L (titer of 2.29 g/L), and about equimolar toiron pyro phosphate at 1.3 g/L (titer of 2.26 g/L).

Basal medium 6.2a is a precursor medium that is almost identical tobasal medium 6.2 except for additionally comprising some non-essentialcofactors and nucleotides and containing no succinic acid, putrescine atonly 4.8 mg/l and a total amino acid concentration of 40 mM instead of44 mM. Further Glutamine was added at a lower amount resulting in aratio relative to isoleucine of 37.42.

In a parallel experiment CHO2 (CHO-DG44) Rituximab cells were culturedin basal medium 6.2a with iron choline citrate at differentconcentrations (0 g/l, 0.2 g/l, 0.4 g/l or 2 g/l) and feed medium 6.2acontaining iron choline citrate at 0.56 g/l or in basal medium 6.2a withiron citrate at 0.1 g/l and feed medium 6.2a containing iron citrate at0.25 g/l. The concentration of iron citrate (0.1 g/l and 0.25 g/l) waschosen to be about equimolar to iron choline citrate at 0.2 g/l in thebasal medium and 0.56 g/l in the feed medium.

Feed medium 6.2a is a precursor medium that is almost identical to feedmedium 6.2 except for additionally comprising some non-essentialcofactors and nucleotides and slightly higher sodium bicarbonate andcontaining putrescine at only 33.02 mg/l and a total amino acidconcentration of 511 mM instead of 508 mM. Further alanine wasadditionally present in the medium with a ratio relative to isoleucineof 0.15.

Shake flasks with a starting volume of 60 ml in 250 ml flasks wereincubated at 37° C. in an incubator (10% CO2 atmosphere was providedfrom day 0 to 3 followed by 3% CO2 for one day and 0% CO2 until the endof the cultivation). Feeding solution was added every day at a feed rateof 30 ml/L/d starting on day two. Glucose was fed on demand to maintainthe actual glucose concentration between 2-4 g/l over the cultivationperiod. Total cells, viable cells, viability, product concentration,glucose concentration, lactic acid concentration, ammonium concentrationand osmolarity were measured every second day until the end ofcultivation to monitor and control the experimental progress.Experiments were performed in duplicates (N=2).

Results:

Product titers with iron choline citrate at 1.0 g/L were significantlyhigher than control at 0.2 g/L iron choline citrate (titer of 2.81 g/Lvs. 2.07 g/L in control) and even slightly higher than product titerswith iron choline citrate at 2.0 g/l (titer of 2.67 g/L) (FIG. 7B).Further, product titers were considerably higher with iron cholinecitrate at 2.0 g/L or 1.0 g/L (titer 2.67 g/L or 2.81 g/L) compared toiron pyro phosphate (titer of 2.24 g/L-2.37 g/L) or iron phosphate(titer of 2.29 g/L-2.38 g/L) at about equimolar amounts (FIG. 7B).

Maximal viable cell concentration was achieved with 2.0 g/L iron cholinecitrate in basal medium resulting in an improved cell cultureperformance compared to control (0.2 g/L iron choline citrate in basalmedium) and to most commonly used iron carriers tested with differentconcentrations (FIG. 7A). The viable cell concentration of cellscultured in a basal medium comprising iron phosphate and ironpyrophosphate as iron carriers sharply declined from day 10 to 11 withnegative impact on cell culture performance.

Similar results were found in a parallel experiment. Product titers withiron choline citrate at 2.0 g/L were higher than negative controlcultures without iron choline citrate (titer of 3.06 g/L vs. 2.19 g/L innegative control) or in cultures with iron choline citrate in the basalmedium at 0.4 g/L (titer of 2.87 g/L) or at 0.2 g/L (titer of 2.66 g/L)(FIG. 7C). At lower iron choline citrate concentrations (<1 g/l) theeffect seemed to be concentration dependent and a considerable increasein product concentration was achieved when iron choline citrate wasadded at 0.2 g/L compared to the negative control without iron cholinecitrate.

Further, product titers with iron citrate at 0.1 g/L were lower thanequimolar iron choline citrate at 0.2 g/L (titer of 2.38 g/L vs. 2.66g/L) (FIG. 7D).

Example 8

The effect of different concentrations of iron choline citrate on cellculture performance, specifically cell growth and product formation, wasinvestigated in shake flask experiments using an RPMI based medium. Itwas found that (i) iron choline citrate increased product titers and(ii) that the novel compound iron choline citrate is superior comparedto commonly used iron carriers such as, iron citrate.

Material and Methods:

The experiment was performed in 250 ml shake flasks with a startingvolume of 60 ml. All cultures were seeded at 0.3×10⁶ cells/ml (CHO2(CHO-DG44) cells producing Rituximab) in basal medium 3.1 containingiron choline citrate at different concentrations (0 g/l, 0.2 g/l, 0.4g/l, or 2 g/l) or iron citrate (0.1 g/I, 0.2 g/l or 1 g/l) at aboutequimolar amounts and feed medium 2 containing iron citrate at 0.25 g/l.The concentration of iron citrate of 0.1 g/l, 0.2 g/l and 1 g/l waschosen to be about equimolar to iron choline citrate at 0.2 g/l, 0.4 g/land 2 g/l in the basal medium, respectively.

Shake flasks were incubated at 37° C. in an incubator (10% CO2atmosphere was provided from day 0 to 3 followed by 5% CO2 for one dayand 0% CO2 until the end of the cultivation). Feeding solution was addedevery day at a feed rate of 30 ml/L/d starting on day two. Glucose wasfed on demand to maintain the actual glucose concentration between 2-4g/l over the cultivation period. Total cells, viable cells, viability,product concentration, glucose concentration, lactic acid concentration,ammonium concentration and osmolarity were measured every second dayuntil the end of cultivation to monitor and control the experimentalprogress. Experiments were performed in duplicates (N=2).

Results:

Product titers with iron choline citrate at 2.0 g/L were higher thannegative control without iron choline citrate (titer of 0.244 g/L vs.0.156 g/L in negative control) or in cultures with iron choline citratein the basal medium at 0.4 g/L (titer of 0.217 g/L) or 0.2 g/L (titer of0.194 g/L) (see FIG. 8D and compare FIGS. 8A, B and C). Thus, the effectof iron choline citrate seemed to be concentration dependent and aconsiderable increase in product concentration was achieved when ironcholine citrate was added at 0.2 g/L compared to the negative controlwithout iron choline citrate.

Further, product titers with iron citrate at 0.1 g/L were lower thanequimolar iron choline citrate at 0.2 g/L (titer of 0.184 g/L vs. 0.194g/L; FIG. 8A). Likewise product titers with iron citrate at 0.2 g/L werelower than equimolar iron choline citrate at 0.4 g/L (titer of 0.200 g/Lvs. 0.217 g/L; FIG. 8B) and product titers with iron citrate at 1.0 g/Lwere significantly lower than equimolar iron choline citrate at 2.0 g/L(titer of 0.201 g/L vs. 0.244 g/L; FIG. 8C).

Overall viable cell concentrations showed similar profiles for ironcholine citrate and iron citrate, but equimolar concentration of ironcholine citrate vs. iron citrate resulted in higher viable cellconcentrations (e.g., titers obtained by equimolar iron citrate with 1.0g/L (titer 201 mg/L) were clearly lower than those obtained with 2.0 g/liron choline citrate (titer 244 mg/L)). Further, compared to commercialiron carriers such as iron citrate, the (equimolar) application of ironcholine citrate resulted in lower osmolarity values, which is consideredto be beneficial for mammalian cell culture with respect to viable cellconcentration and cell viability. Compared to negative control (noaddition of iron choline citrate) iron choline citrate increasedosmolarity only slightly (data not shown). Viable cell concentrationsand cell viability were only slightly improved when iron choline citratewas added in increasing concentrations.

Example 9

The effect of iron choline citrate and equimolar iron citrate in mediaplatform 6.2 or an RPMI based medium platform (basal medium 3.1 and feedmedium 2) on cell culture performance in fed-batch mode in a 2 Lbioreactor, specifically cell growth and product formation, wasinvestigated (CHO2 (CHO-DG44) producing Rituximab). It was found that(i) iron choline citrate increased product titer, (ii) and was superiorcompared to commonly used iron carriers such as iron citrate. Hence, thepositive effects of iron choline were independent of the appliedcultivation system (e.g. shake flask experiments or controlled 2 Lbioreactors or the medium used).

Material and Methods:

The experiment was performed in a fully controlled 2 L bioreactor systemwith a starting volume of 1.8 L. CHO2 (CHO-DG44) Rituximab cells wereseeded at 0.3×10⁶ cells/ml in all cultures using basal medium 6.2acontaining iron choline citrate (0.2 g/l or 2.0 g/l) or iron citrate (1g/l) and feed medium 6.2a containing iron choline citrate at 0.56 g/l(FIGS. 9A-C) or RPMI based basal medium 3.1 containing iron cholinecitrate (0.2 g/l or 2.0 g/l) or iron citrate (1 g/l) and RPMI based feedmedium 2 containing iron citrate at 0.25 g/l. The concentration range ofiron citrate was chosen to be in the same range (on a molar basis) asiron choline citrate. The bioreactors were incubated at 37° C. for theentire cultivation and dissolved CO2 was controlled between 2-15% toprevent toxic concentrations based on the pH set-point of (7.07 on days0-3 and 6.92 on days 3-day 14)+/−0.17. DO set-point was 60% and feed wasadded continuously at 30 ml/L/d. Viable cells, viability, productconcentration, glucose concentration, lactic acid concentration,ammonium concentration and osmolarity were measured as described aboveaccording to the sample intervals. The feed media contained glucose andglucose level was maintained at >2 g/L for the entire cultivation.Glutamine was added from a stock solution on demand to keep theglutamine concentration in the range of 0.1-0.4 g/l. Experiments wereperformed in duplicates (N=2).

Results:

Product titers with iron choline citrate at 2.0 g/L in basal medium 6.2awere higher compared to control cultures with 0.2 g/L iron cholinecitrate (titer of 2.04 g/L vs. 1.62 g/L in control) or 1.0 g/L ironcitrate (titer of 1.73 g/L) (FIG. 9C). This demonstrates that the effectof iron choline citrate on product titer was superior compared to ironcitrate at equimolar concentrations. Whereas product concentrations wereincreased, viable cell concentrations and cell viability using differentconcentrations of iron choline citrate or equimolar concentrations ofiron citrate were comparable. A slightly faster decrease in viable cellconcentration and viability starting on day 8 for cultures treated withiron choline citrate was observed (FIGS. 9A and B). The overallosmolarity in cultures using media platform 6.2a was within anacceptable range for all samples (280-approximately 400 mOsmo/kg, day0-12, data not shown).

Likewise product titers with iron choline citrate at 2.0 g/L in an RPMIbased medium were higher compared to control at 0.2 g/L iron cholinecitrate (titer of 0.257 g/L vs. 0.237 g/L in control) or 0.1 g/l ironcitrate (titer of 0.200 g/L) (FIG. 9D). Whereas product concentrationswere increased, viable cell concentrations and cell viability in theRPMI based media system at different concentrations of iron cholinecitrate or equimolar iron citrate were comparable. The overallosmolarity in cultures using the RPMI based media platform was slightlyincreased (350-440 mOsmo/kg, day 0-12, data not shown).

Example 10

A glutamine synthetase (GS) deficient cell line derived from CHO-K1(CHO-K1 GS) was transfected in order to express and produce Rituximab asan example protein using a glutamine synthetase-based protein expressionsystem. The growth of this CHO-K1 GS cell line producing Rituximab andthe production of Rituximab as an example protein were analysed. It wasfound that the media with the improved amino acid ratios can also beused for a GS-deficient cell line and that the amount of the producedprotein of interest is comparable high to that of other cell lines (seeFIG. 6C).

Material and Methods:

This experiment was performed in a miniaturized bioreactor system with astarting volume of 15 ml. The CHO-K1 GS cell line expressing Rituximabwas seeded at 0.7×10⁶ cells/ml in basal medium 6.2 GS and cultured usingfeed medium 6.2 GS, both with optimized AA ratios. Compared to the basalmedium 6.2 and feed medium 6.2 some minor changes were made:

-   -   Basal medium 6.2 GS: Elimination of glutamine from AA premix        powder (optimized AA ratios) due to GS system, change from        succinic acid 1.5 g/L to disodium succinate 6H₂O 3.43 g/L        formulation and increase of iron choline citrate from 0.2 g/L to        1.8 g/L.    -   Feed medium 6.2 GS: Increase of glucose from 35.4 g/L to 83.4        g/L and change from Succinic acid 5.26 g/L to disodium succinate        6H₂O 12.0 g/l formulation

The therapeutic molecule expressed in CHO-K1 GS cells was Rituximab witha heavy chain having the amino acid sequence of SEQ ID NO: 1 and a lightchain having the amino acid sequence of SEQ ID NO:2. The bioreactorswere incubated at 34.5° C. for the entire cultivation and dissolved CO₂was controlled between 2-15% to prevent toxic concentrations based onthe pH set-point of 6.95 (+/−0.25).

For this fed-batch application a platform process for successfulscale-up was applied that included a standard feed rate of 30 ml/L/d.This means that the nutrient feed solution was added daily for theentire cultivation from day 1 until the end of cultivation. Glucose wasalso added on demand to keep the glucose level >0.6 g/L for the entirecultivation. Viable cells, viability, product concentration, glucoseconcentration, lactic acid concentration, ammonium concentration andosmolarity were measured as described above. Experiments were performedin duplicates (N=2).

Results:

The parameters viable cell density, viability and product concentrationof the duplicates from CHO-K1 GS cell line expressing Rituximab werecomparable or even better compared to other cell lines. The two smallscale bioreactors showed a high preharvest product concentration of 8665mg/L and 8102 mg/L after the 14 days fed batch cultivation process.These results demonstrate that the glutamine synthetase (GS) deficientcell line derived from CHO-K1 was able to proliferate and to produce aprotein of interest at very high titers using the culture medium withthe optimized AA ratios (FIG. 10A-C).

In view of the above, it will be appreciated that the present inventionalso relates to the following items:

Items

-   1. A basal cell culture medium for culturing mammalian cells    comprising the following amino acids at a molar ratio relative to    isoleucine (mM/mM) of:    -   L-leucine/L-isoleucine of about 1.2-2.2,    -   L-phenylalanine/L-isoleucine of about 0.5-0.9,    -   L-tyrosine/L-isoleucine of about 1.5-2.7,    -   L-threonine/I-isoleucine of about 1.0-1.9, and    -   L-valine/L-isoleucine of about 1.0-1.9,    -   wherein the basal cell culture medium has a total amino acid        content of about 25 to 150 mM.-   2. The basal cell culture medium of item 1, further comprising    L-lysine at a molar ratio relative to isoleucine of about 1.6-2.9    (mM/mM).-   3. The basal cell culture medium of items 1 or 2, further comprising    at least one of the following amino acids at a molar ratio relative    to isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.3-0.5,    -   L-proline/L-isoleucine of about 1.6-3.0; or    -   L-methionine/L-isoleucine of about 0.4-0.7.-   4. The basal cell culture medium of item 3, comprising L-tryptophan,    L-proline and L-methionine each at the molar ratios according to    item 3.-   5. A basal cell culture medium for culturing mammalian cells    comprising the following amino acids at a molar ratio relative to    isoleucine (mM/mM) of:    -   L-leucine/L-isoleucine of about 1.3-1.8,    -   L-phenylalanine/L-isoleucine of about 0.6-0.9,    -   L-tyrosine/L-isoleucine of about 1.7-2.5,    -   L-threonine/I-isoleucine of about 1.2-1.8, and    -   L-valine/L-isoleucine of about 1.3-1.6,    -   wherein the basal cell culture medium has a total amino acid        content of about 25 to 100 mM.-   6. The basal cell culture medium of items 1 or 5, further comprising    L-lysine at a molar ratio relative to isoleucine of about 1.8-2.7    (mM/mM).-   7. The basal cell culture medium of items 1, 2, 5 or 6, further    comprising at least one of the following amino acids at a molar    ratio relative to isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.3-0.5,    -   L-proline/L-isoleucine of about 1.6-3.0; or    -   L-methionine/L-isoleucine of about 0.4-0.7.-   8. The basal cell culture medium of item 7, comprising L-tryptophan,    L-proline and L-methionine each at the molar ratios according to    item 7.-   9. The basal cell culture medium of items 5, further comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   (a) L-lysine/L-isoleucine of about 1.8-2.7; and/or    -   (b) L-tryptophan/L-isoleucine of about 0.3-0.5,        -   L-proline/L-isoleucine of about 1.6-3.0; and        -   L-methionine/L-isoleucine of about 0.4-0.7.-   10. A basal cell culture medium for culturing mammalian cells    comprising the following amino acids at a molar ratio relative to    isoleucine (mM/mM) of:    -   L-leucine/L-isoleucine of about 1.5-1.8,    -   L-phenylalanine/L-isoleucine of about 0.6-0.8,    -   L-tyrosine/L-isoleucine of about 1.9-2.3,    -   L-threonine/I-isoleucine of about 1.3-1.6, and    -   L-valine/L-isoleucine of about 1.3-1.6,    -   wherein the basal cell culture medium has a total amino acid        content of about 25 to 100 mM.-   11. The basal cell culture medium of items 1, 5 or 10, further    comprising L-lysine at a molar ratio relative to isoleucine of about    2.0-2.5 (mM/mM).-   12. The basal cell culture medium of items 1, 2, 5, 6, 10 or 11,    further comprising at least one of the following amino acids at a    molar ratio relative to isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.3-0.5,    -   L-proline/L-isoleucine of about 1.6-3.0; or    -   L-methionine/L-isoleucine of about 0.4-0.7.-   13. The basal cell culture medium of item 12, comprising    L-tryptophan, L-proline and L-methionine each at the molar ratios    according to item 12.-   14. The basal cell culture medium of item 10, further comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   (a) L-leucine/L-isoleucine of about 2.0-2.5, and/or    -   (b) L-tryptophan/L-isoleucine of about 0.3-0.5,        -   L-proline/L-isoleucine of about 1.6-3.0; and        -   L-methionine/L-isoleucine of about 0.4-0.7.-   15. The basal cell culture medium of any one of items 1 to 14,    wherein the medium is a serum-free medium, preferably a chemically    defined medium or a chemically defined and protein-free medium.-   16. The basal cell culture medium of any one of items 1 to 15    additionally comprising iron choline citrate at a concentration of    about 0.1 to 5.0 mM, about 0.2 to 2.0 mM, about 0.2 to 1.0 mM or    about 0.4 to 1.0 mM.-   17. The basal cell culture medium of any one of items 1 to 16,    wherein the basal cell culture medium has a total amino acid content    of about 30 to about 80, preferably about 35 to about 65, more    preferably about 40 to about 50 mM.-   18. A basal cell culture medium for culturing mammalian cells    comprising iron choline citrate at a concentration of 0.1 to 5.0 mM,    about 0.2 to 2.0 mM, about 0.2 to 1.0 mM or about 0.4 to 1.0 mM.-   19. A feed medium for culturing mammalian cells comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   L-leucine/L-isoleucine of about 2.3-4.2,    -   L-phenylalanine/L-isoleucine of about 0.6-1.1,    -   L-threonine/I-isoleucine of about 1.3-2.4, and    -   L-valine/L-isoleucine of about 1.1-2.0,    -   wherein the feed medium has a total amino acid content of about        100 to 1000 mM.-   20. The feed medium of item 19, further comprising the following    amino acids at a molar ratio relative to isoleucine (mM/mM) of:    -   L-tyrosine/L-isoleucine of about 0.6-1.1, and/or        L-lysine/L-isoleucine of about 1.1-2.1, preferably    -   L-tyrosine/L-isoleucine of about 0.6-1.1, and        L-lysine/L-isoleucine of about 1.1-2.1.-   21. The feed medium of items 19 or 20, further comprising at least    one of the following amino acids at a molar ratio relative to    isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.3-0.6,    -   L-proline/L-isoleucine of about 0.9-1.8; or    -   L-methionine/L-isoleucine of about 0.4-0.8.-   22. The feed medium of item 21, comprising L-tryptophan, L-proline    and L-methionine each at the molar ratios according to item 21.-   23. A feed medium for culturing mammalian cells comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   L-leucine/L-isoleucine of about 2.6-3.9,    -   L-phenylalanine/L-isoleucine of about 0.7-1.0,    -   L-threonine/I-isoleucine of about 1.5-2.2, and    -   L-valine/L-isoleucine of about 1.3-1.9,    -   wherein the feed medium has a total amino acid content of about        100 to 1000 mM.-   24. The feed medium of items 19 or 23, further comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   L-tyrosine/L-isoleucine of about 0.7-1.0, and/or        L-lysine/L-isoleucine of about 1.3-1.9, preferably    -   L-tyrosine/L-isoleucine of about 0.7-1.0, and        L-lysine/L-isoleucine of about 1.3-1.9.-   25. The feed medium of items 19, 20, 23 or 24, further comprising at    least one of the following amino acids at a molar ratio relative to    isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.4-0.5,    -   L-proline/L-isoleucine of about 1.1-1.6; or    -   L-methionine/L-isoleucine of about 0.5-0.7.-   26. The feed medium of item 25, comprising L-tryptophan, L-proline    and L-methionine each at the molar ratios according to item 25.-   27. The feed medium of item 23, further comprising the following    amino acids at a molar ratio relative to isoleucine (mM/mM) of:    -   (a) L-tyrosine/L-isoleucine of about 0.7-1.0, and        -   L-lysine/L-isoleucine of about 1.3-1.9, and/or    -   (b) L-tryptophan/L-isoleucine of about 0.4-0.5,        -   L-proline/L-isoleucine of about 1.1-1.6; and        -   L-methionine/L-isoleucine of about 0.5-0.7.-   28. A feed medium for culturing mammalian cells comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   L-leucine/L-isoleucine of about 2.9-3.5,    -   L-phenylalanine/L-isoleucine of about 0.8-0.9,    -   L-threonine/I-isoleucine of about 1.7-2.0, and    -   L-valine/L-isoleucine of about 1.4-1.7,    -   wherein the feed medium has a total amino acid content of about        100 to 1000 mM.-   29. The feed medium of items 19, 23 or 28 further comprising the    following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   L-tyrosine/L-isoleucine of about 0.7-0.9, and/or        L-lysine/L-isoleucine of about 1.4-1.8, preferably    -   L-tyrosine/L-isoleucine of about 0.7-0.9, and        L-lysine/L-isoleucine of about 1.4-1.8.-   30. The feed medium of items 19, 20, 23, 24, 28 or 29, further    comprising at least one of the following amino acids at a molar    ratio relative to isoleucine (mM/mM) of:    -   L-tryptophan/L-isoleucine of about 0.4-0.5,    -   L-proline/L-isoleucine of about 1.2-1.5; or    -   L-methionine/L-isoleucine of about 0.5-0.6.-   31. The feed medium of item 30, comprising L-tryptophan, L-proline    and L-methionine each at the molar ratios according to item 30.-   32. The feed medium of item 28, further comprising at least one of    the following amino acids at a molar ratio relative to isoleucine    (mM/mM) of:    -   (a) L-tyrosine/L-isoleucine of about 0.7-0.9, and        -   L-lysine/L-isoleucine of about 1.4-1.8 and/or.    -   (b) L-tryptophan/L-isoleucine of about 0.4-0.5,        -   L-proline/L-isoleucine of about 1.2-1.5; and        -   L-methionine/L-isoleucine of about 0.5-0.6.-   33. The feed medium of any one of items 19 to 32, wherein the feed    medium is a concentrated feed medium for addition to the cell    culture at about 10-50 ml/L/day, preferably at about 15-45 ml/L/day,    more preferably at about 20-40 ml/L/day and more preferably at about    30 ml/L/day based on the culture starting volume.-   34. The feed medium of any one of items 19 to 32, wherein the medium    is a serum-free medium, preferably a chemically defined medium or a    chemically defined and protein-free medium.-   35. The feed medium of any one of items 19 to 34 additionally    comprising iron choline citrate at a concentration of about 0.4 to 5    mM, about 0.4 to 1.0 mM or about 0.5 to 1.0 mM, preferably about 0.5    to 0.6 mM.-   36. The feed medium of any one of items 19 to 35 further    characterized by that it has a low salt content, preferably about    100 mM or less, more preferably 50 mM or less.-   37. The feed medium of any one of items 19 to 36, wherein the feed    medium has a total amino acid content of about 200 to about 900,    preferably about 300 to about 800, more preferably about 400 to    about 700 mM-   38. A feed medium for culturing cells comprising iron choline    citrate at a concentration of about 0.4 to 5 mM, about 0.4 to 1.0 mM    or about 0.5 to 1.0 mM, preferably about 0.5 to 0.6 mM.-   39. A medium platform for culturing mammalian cells comprising:    -   a) the basal cell culture medium of items 1 to 18, and    -   b) the feed medium of items 19 to 38.-   40. The cell culture medium of any one of items 1 to 18 or the feed    medium of any one of items 19 to 38, wherein the mammalian cell is a    rodent or human cell, wherein the rodent cell is preferably a    Chinese hamster ovary (CHO) cell such as a CHO-K1 cell, a CHO-DG44    cell, a DuxB11 cell or a CHO GS deficient cell, most preferably the    cell is a CHO-DG44 cell or a CHO GS deficient cell.-   41. A method of generating a basal cell culture medium comprising:    -   a) providing a basal cell culture medium, and    -   b) adding amino acids at or adjusting the amino acid ratios to        the final molar ratio according to items 1 to 17.-   42. The method of item 41, further comprising a step of adding or    adjusting as an iron source iron choline citrate at a concentration    of about 0.1 to 5.0 mM, about 0.2 to 2.0 mM, about 0.2 to 1.0 mM, or    about 0.4 to 1.0 mM.-   43. A method of generating a feed medium comprising:    -   a) providing a feed medium, and    -   b) adding amino acids at or adjusting the amino acid ratios to        the final molar ratio according to items 19 to 37.-   44. The method of item 43, further comprising a step of adding or    adjusting as an iron source iron choline citrate at a concentration    of about 0.4 to 5 mM, about 0.4 to 1.0 mM or about 0.5 to 1.0 mM,    preferably about 0.5 to 0.6 mM.-   45. The method of any one of items 41 to 44, wherein the medium is a    serum-free medium, preferably a chemically defined medium or a    chemically defined and protein-free medium.-   46. A method of culturing a mammalian cell comprising the following    steps:    -   a) providing mammalian cells,    -   b) culturing the cells in the basal cell culture medium of any        one of items 1 to 18, and    -   c) optionally adding the feed medium of any one of items 19 to        38 to the basal cell culture medium;    -   wherein the cells are cultured under conditions that allow the        cells to proliferate.-   47. A method of producing a protein of interest comprising the    following steps:    -   a) providing mammalian cells comprising a gene of interest        encoding the protein of interest,    -   b) culturing the cells in the basal cell culture medium of any        one of items 1 to 18, and    -   c) optionally adding the feed medium of any one of items 19 to        38 to the basal cell culture medium, and    -   d) optionally separating and/or isolating and/or purifying said        protein of interest from the cell culture;    -   wherein the cells are cultured under conditions that allow        expression of the protein of interest.-   48. The method of item 47, wherein the protein of interest is a    secreted protein, preferably the protein of interest is an antibody    or Fc-fusion protein.-   49. The method of any one of items 46 to 48, wherein the mammalian    cell is a rodent or human cell, preferably the rodent cell is a    Chinese hamster ovary (CHO) cell such as a CHO-K1 cell, a CHO-DG44    cell, a DuxB11 cell or a CHO GS deficient cell, most preferably the    cell is a CHO-DG44 cell or a CHO GS deficient cell.-   50. The method of any one of items 46 to 49, wherein the feed medium    of any one of items 19 to 38 is added to the cells cultured in the    basal cell culture medium and wherein    -   (a) the feed medium is added at about 10-50 ml/L/day, preferably        at about 15-45 ml/L/day, more preferably at about 20-40 ml/L/day        and more preferably at about 30 ml/L/day based on the culture        starting volume to the basal cell culture medium,    -   (b) the feed medium is added starting on day 0, 1, 2 or 3,        and/or    -   (c) the feed medium is added continuously or as a bolus several        times a day, two times a day, once per day, every second day or        every third day.-   51. The method of any one of items 46 to 50, wherein the cell    culture is a large-scale cell culture, preferably a cell culture of    a working volume of 100 L or more, more preferably of 1000 L or more    or even more preferably of 10000 L or more.-   52. A kit of parts comprising the basal cell culture medium of any    one of items 1 to 18 and/or the feed medium of any one of items 19    to 39, and optionally a mammalian cell.-   53. Use of the basal cell culture medium of any one of items 1 to 18    for producing a protein comprising culturing mammalian cells that    produce a protein of interest in said medium for a period of time    and conditions suitable for cell growth and protein production,    harvesting the protein of interest and recovering the protein from    the culture medium or cell lysate.-   54. The use of item 53, further comprising feeding the cells with    the feed medium of any one of items 19 to 38 during said culture    period.-   55. Use of the feed medium of any one of items 19 to 39 for    producing a protein comprising culturing mammalian cells that    produce the protein of interest in the basal cell culture medium of    any one of items 1 to 18 for a period of time and conditions    suitable for cell growth and protein production, feeding the cells    with said feed medium, harvesting the protein of interest and    recovering the protein from the culture medium.-   56. Use of iron choline citrate as iron carrier in a mammalian cell    culture medium, wherein the iron choline citrate is present in the    mammalian cell culture medium at a concentration of about 0.2 to 2.0    mM.

1-4. (canceled)
 5. A basal cell culture medium for culturing mammaliancells comprising iron choline citrate at a concentration of 0.1 to 5.0mM, about 0.2 to 2.0 mM, about 0.2 to 1.0 mM or about 0.4 to 1.0 mM.6-9. (canceled)
 10. A feed medium for culturing cells comprising ironcholine citrate at a concentration of about 0.4 to 5 mM, about 0.4 to1.0 mM or about 0.5 to 1.0 mM, preferably about 0.5 to 0.6 mM. 11-15.(canceled)